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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Cell culture alters Ca²⁺ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells

¹Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; and ²Smooth Muscle Research Centre, Regional Development Centre, Dundalk Institute of Technology, Dundalk, Ireland

Submitted 15 June 2007 ; accepted in final form 30 October 2007

	ABSTRACT

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Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP₃)- and ryanodine-sensitive Ca²⁺ stores activates capacitative Ca²⁺ entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca²⁺ stores and alters Ca²⁺ entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-loaded cells. Mn²⁺ quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15–18 mmHg. In acutely isolated PASMCs, depletion of IP₃-sensitive Ca²⁺ stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca²⁺ transients but abolished 5-HT-induced Ca²⁺ transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca²⁺ transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca²⁺]_i. The transient rise in [Ca²⁺]_i was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca²⁺]_i was inhibited by KB-R7943, a selective inhibitor of reverse mode Na⁺/Ca²⁺ exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca²⁺]_i was inhibited by SKF-96365, Ni²⁺, La³⁺, and Gd³⁺. In addition, store depletion or hypoxia increased the rate of Mn²⁺ quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP₃ and ryanodine receptors into a common intracellular Ca²⁺ compartment, and depletion of this store or hypoxia activates voltage-operated Ca²⁺ entry, reverse mode NCX, and CCE.

capacitative calcium entry; hypoxia; cultured pulmonary artery smooth muscle cells

It is well established that vascular smooth muscle cells undergo phenotypic modulation during in vitro culture. They undergo a transition from a contractile phenotype to a proliferative or synthetic phenotype when they proliferate (see Refs. 4 and 25 for review). Unlike freshly isolated cells, vascular smooth muscle cells maintained in culture may lose their ability to contract because of the lower volume fraction of myofilaments and higher proportion of synthetic organelles (4, 25). These cultured vascular smooth muscle cells with the modified synthetic phenotype resemble cells during response to vascular injury such as atherosclerosis or restenosis (25, 31). Several studies in rat aortic smooth muscle cells have shown that the expression of L-type Ca²⁺ channels decreases during the cell culture process (11, 16, 19). In cultured human PASMCs, although ATP-sensitive K⁺ channels and the properties of these channels in culture are similar to that in other acutely isolated PASMCs from other species, the activity of these channels is suppressed during proliferation (7). In addition, TRPC1 channels were upregulated, and enhanced CCE was observed in human PASMCs during proliferation (13). Furthermore, although IP₃ receptor expression is maintained (36, 40, 45), expression of ryanodine receptors and SERCA may be lost during cell proliferation (40), indicating modifications of SR Ca²⁺ stores. However, the question of whether cell culture changes the organization of the IP₃- and ryanodine-sensitive Ca²⁺ stores and alters Ca²⁺ entry pathways activated by store depletion remains to be demonstrated.

We have previously shown in acutely isolated canine PASMCs that the IP₃-sensitive and ryanodine-sensitive Ca²⁺ stores are functionally independent (17), and depletion of both stores is required to activate CCE (48). We also found that hypoxia causes mobilization of the intracellular stores leading to the activation of VOCC and CCE in these cells (23). The aims of the present study were to utilize the same experimental approaches to determine if cell culture modifies SR Ca²⁺ stores and alters the Ca²⁺ entry pathways activated by store depletion or hypoxia in cultured canine PASMCs.

	MATERIALS AND METHODS

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PASMCs isolation and cell culture. PASMCs were isolated from canine pulmonary artery as previously described (48). Mongrel dogs of either sex were killed with pentobarbital sodium (45 mg/kg iv) and ketamine (15 mg/kg iv), as approved by the University of Nevada at Reno Institutional Care and Use Committee. The heart and lungs were removed en bloc, and the third and fourth branches of the pulmonary artery were dissected in a low-Ca²⁺ physiological salt solution (PSS) composed of the following (in mM): 125 NaCl, 5.36 KCl, 0.34 Na₂HPO₄, 0.44 K₂HPO₄, 1.2 MgCl₂, 11 HEPES, 10 glucose, and 0.05 CaCl₂ (pH 7.4 adjusted with Tris). Arteries were cleaned of connective tissue, cut into small pieces, and placed in a tube containing fresh low-Ca²⁺ PSS. Tissue was immediately digested or cold stored at 5^°C for up to 24 h. To disperse cells, pulmonary arterial tissue was incubated with the low-Ca²⁺ PSS containing (in mg/ml): 0.5 collagenase type XI, 0.04 elastase type IV, and 0.5 BSA (fat free) for 16–18 h at 5°C. The tissue was then transferred to an enzyme-free, low-Ca²⁺ PSS and triturated with a fire-polished Pasteur pipette. The resulting dispersed PASMCs were stored at 5°C and used in experiments within 5–8 h or subjected to cell culture as previously described (8). Freshly dispersed PASMCs were plated on 25-cm² cell cultured flasks and incubated with medium 199 cell culture medium containing 10% newborn calf serum (NCS), 2 mM glutamine, 200 U/ml penicillin, and 200 µg/ml streptomycin. Cells were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C and grown to 90–95% confluence. These primary cells were then trypsinized, passaged on a cover slip, and grown to 70–80% confluence. Confluent cells were then growth arrested in 0.3% NCS medium for 24 h before experimental use.

Morphological and functional comparison between acutely isolated and cultured canine PASMCs. To study the morphological differences between acutely isolated and cultured canine PASMCs, we compare the expression of smooth muscle -actin in these cells using the immunostaining method. The acutely isolated and cultured canine PASMCs were fixed in 4% paraformaldehyde and stained with specific monoclonal antibody raised against smooth muscle -actin (1:300; Sigma, St. Louis, MO). A secondary antibody conjugated with Alexa Flour 488 (1:200; Molecular Probes) was used to display the fluorescence image (excited at 495 nm and emitted at 519 nm). The cells were then mounted in Vectashield mounting medium containing propidium iodide (Vector Laboratories). Propidium iodide was used to stain cell nuclei and was excited at 535 nm and emitted at 615 nm to visualize the cell nuclei. The cells were examined under a Bio-Rad Radiance 2100 inverted laser-scanning confocal microscope with a Nikon Plan-Fluor x40 oil immersion objective. For control experiments, the cells were treated similarly in the absence of primary antibody. To study the functional differences between the acutely isolated and cultured canine PASMCs, the cells were imaged under control conditions and then subjected to 5 min exposure to 10 µM 5-HT, a potent pulmonary vasoconstrictor. After exposures of cells to 5-HT, images of these cells were taken again, and the contractility of these cells was then compared between the acutely isolated and cultured canine PASMCs.

Measurement of intracellular Ca²⁺. [Ca²⁺]_i was estimated in PASMCs loaded with fura 2-AM (Molecular Probes, Eugene, OR) using a dual excitation digital Ca²⁺ imaging system (IonOptix, Milton, MA) equipped with an intensified charge-coupled device (CCD) camera as previously described (48). PASMCs were loaded with 10 µM fura 2-AM for 30 min in the dark at room temperature and placed on the cover slip in a 0.2-ml perfusion chamber mounted on an inverted epifluorescence microscope (Nikon) outfitted with a x40 oil immersion objective (NA1.3; Nikon). Cells were then washed several times at 1 ml/min to remove extracellular fura 2-AM with 2 mM Ca²⁺-PSS composed of the following (in mM): 113 NaCl, 5 KCl, 1 MgCl, 2 CaCl₂, 0.5 NaH₂PO₄, 24 NaHCO₃, and 10 glucose (gassed continuously with 21% O₂-5% CO₂-74% N₂, pH 7.4). Cells were illuminated with a xenon arc lamp at 340 ± 15 and 380 ± 12 nm (Omega Optical, Brattleboro, VT), and emitted light was collected from regions that encompassed single cells with a CCD camera at 510 nm (Nikon). All experiments were performed at 35–37°C, and images were acquired at 1 Hz and stored on compact disk for later analysis. Background fluorescence was collected automatically and subtracted from the acquired fluorescence video images during each experiment. The ratio of fluorescence (R) excited at the two excitation wavelengths was used to estimate [Ca²⁺]_i as described by Grynkiewicz et al. (14):

The values for Sf₂ (fluorescence measured at 380 nm in Ca²⁺-free solution), Sb₂ (fluorescence measured at 380 nm in Ca²⁺-saturating conditions), R_min (minimum ratio), and R_max (maximum ratio) were determined from in situ calibrations of fura 2 for each cell. The dissociation constant for Ca²⁺ binding (K_d) was assumed to be 224 nM (14). To determine R_min, cells were dialyzed with 4 µM ionomycin in Ca²⁺-free PSS containing 10 mM EGTA at the end of each experiment. R_max was determined from cells dialyzed with 4 µM ionomycin in PSS containing 10 mM CaCl_2.

In experiments where the organization of the SR Ca²⁺ stores was studied, the experiments were performed in 2 mM Ca²⁺-PSS. In experiments where the effects of store depletion were investigated, CPA and brief exposure to 5-HT were used to deplete IP₃-sensitive Ca²⁺ stores, whereas 10 µM ryanodine and brief exposure to caffeine were used to deplete ryanodine-sensitive Ca²⁺ stores. Both SR Ca²⁺ stores were maximally depleted by exposure of cells to a cocktail containing 10 µM CPA and 10 µM ryanodine followed by a 30-s exposure to 10 µM 5-HT and 10 mM caffeine in Ca²⁺-free solutions as previously described (48). In the continued presence of CPA and ryanodine, cells were incubated in Ca²⁺-free PSS for 10 min followed by reexposure of cells with 2 mM Ca²⁺-PSS for another 10–15 min. Ca²⁺-free PSS was identical to 2 mM Ca²⁺-PSS but with CaCl₂ omitted and 1 mM EGTA added. An elevation in [Ca²⁺]_i above basal levels during 2 mM Ca²⁺ readdition was used as a marker of CCE-mediated extracellular Ca²⁺ entry. In experiments where the Ca²⁺ influx through SOCs was studied, the rate of Mn²⁺-induced quenching of fura 2 fluorescence was recorded during excitation at 360 nm in nominally Ca²⁺-free PSS containing 10 µM nifedipine (48). Nominally Ca²⁺-free solutions were similar to Ca²⁺-free PSS but with EGTA omitted to avoid chelation of Mn²⁺. In experiments where the effects of LaCl₃ and GdCl₃ were studied, a bicarbonate-, EGTA-, and phosphate-free HEPES-PSS was used to avoid precipitation and chelation of La³⁺ and Gd³⁺ (30, 34, 43, 44).

In experiments where the effect of hypoxia was investigated, hypoxia was induced by switching normoxic PSS to hypoxic PSS, which continuously superfused the cells in the recording chamber as previously described (23). Hypoxic PSS was prepared by continuous gassing with uncertified gas mixture containing 95% N₂ and 5% CO₂ (Sierra Welding, Sparks, NV). The uncertified gas mixture contained a minimal amount of oxygen, which equilibrated with PSS to avoid exposure of cells to anoxic conditions. All solutions were placed in a water bath at 37°C, saturated with either normoxic or hypoxic gas mixtures for at least 30 min before the start of perfusion, and maintained at pH 7.4. The PO₂, measured in preliminary experiments with an O₂-sensitive electrode (MI-730; Microelectrodes, Bedford, NH), was 145 ± 1 mmHg during normoxic PSS perfusion and fell to 15 ± 1 mmHg within 79 ± 2 s of hypoxic exposure. The PO₂ of hypoxic solutions was measured at the end of each experiment and was found to be 15–18 mmHg, ensuring that the PO₂ did not approach anoxia during recording of each experiment.

Drug solutions and data analysis. CPA, KB-R7943, nifedipine, and ionomycin were dissolved in dimethyl sulfoxide. Other drugs were dissolved in deionized water. Data are expressed as means ± SE of n acutely isolated or cultured cells from at least three dogs. Statistical comparisons employed Student's paired or unpaired t-tests or one-way ANOVA with Tukey's pairwise comparison as appropriate. A value of P < 0.05 was considered significant.

	RESULTS

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Cell culture alters the expression of smooth muscle -actin and the contractile function in canine PASMCs. To study if cell culture causes morphological changes in canine PASMCs, we compared the expression of smooth muscle -actin in acutely isolated and cultured PASMCs. We found that the expression of -actin in acutely isolated cells (Fig. 1A) was significantly higher than in cultured cells (Fig. 1B). To determine if the loss of -actin during cell culture correlates with the loss in smooth muscle cell contractile function, we compared the effects of 5-HT in acutely isolated and cultured canine PASMCs. Figure 1C shows the relaxed acutely isolated cells before administration of 5-HT, and most cells were contracted following exposure to 10 µM of 5-HT (Fig. 1D). In contrast, exposure of cultured cells to 10 µM of 5-HT produced only little or no contraction (Fig. 1F) compared with the control (Fig. 1E).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Cell culture alters smooth muscle -actin expression and contractile function in canine pulmonary artery smooth muscle cells (PASMCs). A: expression of smooth muscle -actin in acutely isolated PASMC (bar = 10 µm). B: expression of smooth muscle -actin in cultured PASMC (bar = 10 µm). C: image of acutely isolated PASMCs under resting conditions. D: image of acutely isolated PASMCs in C after 5 min exposure to 10 µM 5-HT (bar = 20 µm). E: image of cultured PASMCs under resting conditions. F: image of cultured PASMCs in E after 5 min exposure to 10 µM 5-HT (bar = 20 µm).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Cell culture modifies intracellular Ca2+ stores in canine PASMCs. A: effect of 10 µM cyclopiazonic acid (CPA) in acutely isolated PASMCs. B: effect of 10 µM CPA in cultured PASMCs. C: effects of transient exposures of 10 mM caffeine and 10 µM 5-HT in the presence of 10 µM CPA in acutely isolated PASMCs. D: bar graph showing mean changes in Ca2+ transients in acutely isolated PASMCs elicited by caffeine (open bar, n = 97) and 5-HT (filled bar, n = 73) as well as the mean changes in Ca2+ transients caused by the first (1) and second (2) brief caffeine (first, n = 53; second, n = 53) and 5-HT (first, n = 75; second, n = 75) exposures in the presence of CPA. **P < 0.01 (ANOVA). E: effects of transient exposures of 10 mM caffeine and 10 µM 5-HT in the presence of 10 µM CPA in cultured PASMCs. F: bar graph showing mean changes in Ca2+ transients in cultured PASMCs elicited by caffeine (open bar, n = 197) and 5-HT (filled bar, n = 136) as well as the mean changes in Ca2+ transients caused by the first (1) and second (2) brief caffeine (first, n = 174; second, n = 50) and 5-HT (first, n = 130; second, n = 77) exposures in the presence of CPA. **P < 0.01 (ANOVA).

Store depletion causes activation of VOCC, reverse-mode NCX, and CCE in cultured canine PASMCs. To study Ca²⁺ entry pathway(s) activated by store depletion in cultured PASMCs, control experiments were first performed in Ca²⁺-free solutions followed by readdition of 2 mM Ca²⁺ (Fig. 3A). Removal of extracellular Ca²⁺ caused a decrease in [Ca²⁺]_i from a basal level of 244 ± 21 nM (R = 0.70 ± 0.02, n = 115) to 41 ± 8 nM (R = 0.44 ± 0.02, n = 115). Subsequent addition of 2 mM Ca²⁺ elicited a very small transient rise in [Ca²⁺]_i, 77 ± 13 nM (R = 0.06 ± 0.01) above basal levels (n = 115, P < 0.01; Fig. 3, A and C), which decayed slowly to the baseline. Figure 3B shows that, when cells were superfused with Ca²⁺-free PSS containing CPA, ryanodine, 5-HT, and caffeine, an early transient increase in [Ca²⁺]_i was observed, indicating Ca²⁺ release from the intracellular stores. This early transient rise in [Ca²⁺]_i decayed slowly to a mean level (16 ± 3 nM, R = 0.44 ± 0.01, n = 171, P < 0.01) below baseline (243 ± 11 nM, R = 0.69 ± 0.01). Subsequent addition of 2 mM Ca²⁺ in the presence of CPA and ryanodine elicited a significant transient rise in [Ca²⁺]_i of 374 ± 33 nM (R = 0.15 ± 0.01) followed by a sustained rise in [Ca²⁺]_i of 76 ± 7 nM (R = 0.047 ± 0.003) above basal levels (n = 171, P < 0.01; Fig. 3C). Part of the transient rise in [Ca²⁺]_i was mediated by Ca²⁺ influx through VOCCs because nifedipine significantly reduced the rise in [Ca²⁺]_i to 213 ± 11 nM (R = 0.15 ± 0.01, n = 347, P < 0.01; Fig. 3, B and D), a concentration (10 µM) causing maximal inhibition of these channels in canine PASMCs (10). However, nifedipine did not affect the sustained rise in [Ca²⁺]_i (Fig. 3, B and D).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Store depletion increases intracellular Ca2+ concentration ([Ca2+]i) in cultured canine PASMCs. A: effects of external Ca2+ removal on fura 2 fluorescence ratio during 2 mM Ca2+ readdition. B: effects of external Ca2+ removal, 10 µM CPA, 10 µM ryanodine, and brief exposures to 10 µM 5-HT and 10 mM caffeine on fura 2 fluorescence ratio during 2 mM Ca2+ readdition, in the absence and presence of 10 µM nifedipine. C: mean changes in [Ca2+]i compared with the resting [Ca2+]i in control (n = 115) and store depletion (n = 171) experiments. Open bars indicate mean decrease in [Ca2+]i. Shaded and filled bars indicate mean transient and sustained rise in [Ca2+]i, respectively. **P < 0.01 and ++P < 0.01 (ANOVA). D: bar graph showing mean changes in transient and sustained rise in [Ca2+]i caused by store depletion after readdition of 2 mM Ca2+ in the absence (open bars, n = 171) and presence (filled bar, n = 347) of 10 µM nifedipine. **P < 0.01 (Student's unpaired t-test).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Store depletion activates reverse-mode Na+/Ca2+ exchanger (NCX) and capacitative Ca2+ entry (CCE) in cultured canine PASMCs. A: 10 and 50 µM KB-R7943 reduced store depletion-activated rise in fura 2 fluorescence ratio in the presence of 10 µM nifedipine. B: bar graph showing mean changes in nifedipine-insensitive transient and sustained rise in [Ca2+]i in the absence (n = 347) and presence of 10 µM (n = 56) and 50 µM (n = 43) KB-R7943. **P < 0.01 (Student's unpaired t-test). C: 500 µM Ni2+ and 100 µM La3+ abolished store depletion-activated sustained rise in fura 2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes of the nifedipine-insensitive sustained rise in [Ca2+]i in the absence (n = 347) and presence (n = 41) of 50 µM SKF-96365, 500 µM Ni2+ (n = 15), 100 µM La3+ (n = 50), and 100 µM Gd3+ (n = 38). **P < 0.01 (ANOVA).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive or ryanodine-sensitive Ca2+ stores alone activates CCE in cultured canine PASMCs. A: changes in fluorescence intensity (arbitrary units) were continuously recorded in nominally Ca2+-free solution, followed by addition of 30 µM MnCl2 and 10 µM nifedipine. Depletion of both IP3- and ryanodine-sensitive Ca2+ stores with sustained exposure to CPA (10 µM) and ryanodine (10 µM) and transient exposures to 5-HT (10 µM) and caffeine (10 mM) increased the rate of Mn2+ quench of fura 2 fluorescence in the presence of 10 µM nifedipine. B: depletion of IP3-sensitive Ca2+ stores alone with 10 µM CPA and 10 µM 5-HT increased the rate of Mn2+ quench of fura 2 fluorescence. C: depletion of ryanodine-sensitive Ca2+ stores alone with 10 µM ryanodine and 10 mM caffeine increased the rate of Mn2+ quench of fura 2 fluorescence. D: bar graph showing percentage change in fura 2 quench after store depletion in the absence (n = 148) and presence (n = 67) of 500 µM Ni2+, 50 µM SKF-96365 (n = 92), 100 µM La3+ (n = 67), and 100 µM Gd3+ (n = 49). **P < 0.01 (ANOVA).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Hypoxia increases [Ca2+]i in cultured canine PASMCs. A: effects of normoxia and external Ca2+ removal on fura 2 fluorescence ratio during 2 mM Ca2+ readdition in the absence and presence of 10 µM nifedipine. B: effects of hypoxia and external Ca2+ removal on fura 2 fluorescence ratio during 2 mM Ca2+ readdition. C: mean changes in [Ca2+]i compared with the resting [Ca2+]i in control (n = 117) and hypoxia (n = 136) experiments. Open bars indicate mean decrease in [Ca2+]i. Shaded and filled bars indicate mean transient and sustained rise in [Ca2+]i, respectively. **P < 0.01 and ++P < 0.01 (ANOVA). D: bar graph showing mean changes in transient and sustained rise in [Ca2+]i caused by hypoxia after readdition of 2 mM Ca2+ in the absence (open bars, n = 136) and presence (filled bar, n = 338) of 10 µM nifedipine. **P < 0.01 (Student's unpaired t-test).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Hypoxia activates reverse-mode NCX and CCE in cultured canine PASMCs. A: 10 and 50 µM KB-R7943 reduced the hypoxia-activated rise in fura 2 fluorescence ratio in the presence of 10 µM nifedipine. B: bar graph showing mean changes in hypoxia-induced nifedipine-insensitive transient and sustained rise in [Ca2+]i in the absence (n = 338) and presence (n = 65) of 10 and 50 µM KB-R7943 (n = 43). *P < 0.05 and **P < 0.01 (Student's unpaired t-test). C: 500 µM Ni2+ and 100 µM La3+ abolished hypoxia-induced sustained rise in fura 2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes of hypoxia-induced nifedipine-insensitive sustained rise in [Ca2+]i in the absence (n = 338) and presence (n = 84) of 50 µM SKF-96365, 500 µM Ni2+ (n = 102), 100 µM La3+ (n = 80), and 100 µM Gd3+ (n = 133). **P < 0.01 (ANOVA).

View larger version (11K): [in this window] [in a new window]   Fig. 8. Hypoxia increases the rate of Mn2+ quench of fura 2 fluorescence in cultured canine PASMCs. A: changes in fluorescence intensity (arbitrary units) were continuously recorded in nominally Ca2+-free solution, followed by addition of 30 µM MnCl2 and 10 µM nifedipine, in normoxic and hypoxic solutions as indicated. Hypoxia increased the rate of Mn2+ quench of fura 2 fluorescence in the presence of 10 µM nifedipine. B: bar graph showing percentage change in fura 2 quench after exposure of cells to hypoxic solution in the absence (n = 112) and presence (n = 35) of 500 µM Ni2+, 50 µM SKF-96365 (n = 27), 100 µM La3+ (n = 70), and 100 µM Gd3+ (n = 65). **P < 0.01 (ANOVA).

	DISCUSSION

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The modification of the IP₃-sensitive and ryanodine-sensitive Ca²⁺ stores found in the present study may be explained by cell proliferation during cell culture. Several studies have shown that cell proliferation affects the expression levels and functions of IP₃ receptors (6, 36) and ryanodine receptors (20, 36, 40) in cultured vascular smooth muscle cells. In spontaneous hypertensive rat aorta myocytes, the ryanodine-sensitive Ca²⁺ stores and thapsigargin-sensitive Ca²⁺ stores were impaired in proliferating cultures (6). In addition, proliferation of rat arterial smooth muscle cells, induced by serum or growth factor, resulted in the disappearance of ryanodine receptors and SERCA2a and in the loss of the caffeine- and ryanodine-sensitive Ca²⁺ stores (40). However, the expression of ryanodine receptors and SERCA2a was maintained in low-serum, nonproliferative conditions (40). A similar study in rat cultured aorta smooth muscle cells showed that caffeine-induced Ca²⁺ release was reduced in the proliferating phase, but growth arresting the cells in serum-free medium restored the sensitivity of cells to caffeine (20). Hence, perhaps the functionally separated IP₃ and ryanodine receptors in acutely isolated canine PASMCs disappear during the cell culture proliferative state, and, when the cells were subjected to subculture and growth arrested in low-serum medium, the receptors reappear but are reorganized into a single compartment.

In our previous study, store depletion activated only CCE (48), whereas hypoxia activated VOCCs and CCE in acutely isolated canine PASMCs (23). Interestingly, we have identified three distinct Ca²⁺ components activated by store depletion and hypoxia in cultured canine PASMCs. Following store depletion or hypoxia in Ca²⁺-free conditions, a transient rise in [Ca²⁺]_i was activated after readmission of 2 mM Ca²⁺, which was partially inhibited by 10 µM nifedipine (Figs. 3B and 6B), suggesting that the Ca²⁺ entry process was mediated at least in part through VOCCs. This is also known to occur in cultured rat PASMCs (21). The opening of VOCCs may be because of Ca²⁺ inhibition of voltage-gated K⁺ channels, leading to membrane depolarization and subsequent activation of VOCCs (29). It is also possible that Ca²⁺ release from stores may activate Ca²⁺-dependent Cl^– channels, leading to membrane depolarization and hence activation of VOCCs (5, 22). The latter mechanism may be more apparent in cultured canine PASMCs because VOCCs are activated in cultured cells but not in acutely isolated cells. This could be explained by the possibility that, during cell culture, IP₃- and ryanodine-sensitive Ca²⁺ stores are reorganized into a common compartment that has a higher Ca²⁺ content as described above. It is very likely that the higher rise in [Ca²⁺]_i induced by store depletion in cultured cells causes increased opening of Ca²⁺-activated chloride channels, leading to more depolarization of membrane potential, hence more VOCC activation.

The transient rise in [Ca²⁺]_i was partially inhibited by nifedipine, leaving a nifedipine-insensitive transient and nifedipine-insensitive sustained rise in [Ca²⁺]_i. Interestingly, the dihydropyridine-insensitive transient rise in [Ca²⁺]_i was inhibited by KB-R7943, suggesting Ca²⁺ entry through reverse-mode NCX. In addition, the transient rise in [Ca²⁺]_i was also inhibited by SKF-96365, Ni²⁺, La³⁺, and Gd³⁺. These blockers are also known to inhibit NCX (e.g., Refs. 24, 28, 33, and 39 and see Ref. 3 for review). This finding is interesting because activation of reverse-mode NCX by store depletion or hypoxia has not been reported in acutely isolated pulmonary artery preparations (e.g., Refs. 22, 23, 30, 44, 48). It is very likely that NCX is upregulated during cell culture (50) and thereby contributes to the enhanced Ca²⁺ entry after cells are subjected to store depletion or hypoxia. Thus store depletion or hypoxia causes activation of store-operated nonselective cation channels, leading to Na⁺ and Ca²⁺ entry. The resultant increase in intracellular Na⁺ concentration may activate the operation of NCX in the reverse mode and subsequently cause more Ca²⁺ entry from the extracellular space. A similar finding is recently reported in human cultured PASMCs (49, 50) and tracheal smooth muscle cells (15), in which Ca²⁺ entry through reverse-mode NCX plays an important role in refilling of intracellular Ca²⁺ stores.

The nifedipine-insensitive sustained rise in [Ca²⁺]_i was abolished by SKF-96365, Ni²⁺, La³⁺, and Gd³⁺. This pharmacological property is similar to CCE, as previously described (30, 34, 43, 44). In addition, store depletion or hypoxia accelerated Mn²⁺ quench of fura 2 fluorescence at a rate similar to that found for store depletion-activated Mn²⁺ entry in acutely isolated canine PASMCs (23, 48). Furthermore, the increases in fura 2 quench rates induced by store depletion or hypoxia in cultured canine PASMCs were both inhibited by SKF-96365, Ni²⁺, La³⁺, and Gd³⁺, confirming that the dihydropyridine-insensitive rise in [Ca²⁺]_i was mediated through a pathway similar to CCE. It is noteworthy that Mn²⁺ is also known to be a putative blocker of NCX (10, 39, see Ref. 3 for review). Thus we do not examine the effects of KB-R7943 on Mn²⁺ quench rate because it is unlikely that Mn²⁺ can enter the cytosol through NCX to quench fura 2 fluorescence. Therefore, the increase in Mn²⁺ quench rate induced by store depletion (Fig. 5) or hypoxia (Fig. 8) is because of Mn²⁺ entry through SOCs and not NCX. The pharmacological properties of CCE in cultured canine PASMCs described here are identical to those of CCE in acutely isolated canine PASMCs, when experiments were performed in phosphate-free HEPES-PSS (data not shown). These properties of CCE are consistent with other studies reported by Robertson et al. (30), Snetkov et al. (34), and Wang et al. (43, 44) in pulmonary arteries.

In conclusion, our present study provides the first direct evidence that cell culture modifies intracellular Ca²⁺ stores, in which the IP₃ and ryanodine receptors are possibly reorganized in the same intracellular Ca²⁺ compartment in canine PASMCs and depletion of this store activates VOCCs, reverse-mode NCX, and CCE. The recruitment of these Ca²⁺ entry pathways may be important for PASMC proliferation during cell culture and a trigger for hypoxic pulmonary vasoconstriction. However, the molecular signals that cause the changes in cell phenotype during cell culture remain to be elucidated. Our present findings in cultured PASMCs may serve as an important pathophysiological model to study pulmonary vascular diseases, and the molecular signals that cause the changes in cell phenotype may be useful targets for the development of new drugs to treat pulmonary hypertension.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants HL-49254 and P20RR-15581.

	ACKNOWLEDGMENTS

 
We thank Hong-Lin Tian and Phillip Keller for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. R. Hume, Dept. of Pharmacology/318, Univ. of Nevada School of Medicine, 1664 North Virginia St., Reno, NV 89557 (e-mail: joeh{at}med.unr.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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Albert AP, Large WA. Activation of store-operated channels by noradrenaline via protein kinace C in rabbit portal vein myocytes. J Physiol (Lond) 544: 113–125, 2002.[Abstract/Free Full Text]

2. Barritt GJ. Receptor activated Ca²⁺ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca²⁺ signaling requirements. Biochem J 337: 153–169, 1999.[CrossRef][Web of Science][Medline]

3. Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev 79: 763–854.

4. Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 59: 1–61, 1979.[Free Full Text]

5. Clapp L, Turner JL, Kozlowski RZ. Ca²⁺-activated Cl^– currents in pulmonary arterial myocytes. Am J Physiol Heart Circ Physiol 270: H1577–H1584, 1996.[Abstract/Free Full Text]

6. Côrtes SF, Lemos VS, Stoclet JC. Alterations in calcium stores in aortic myocytes from spontaneously hypertensive rats. Hypertension 29: 1322–1328, 1997.[Abstract/Free Full Text]

7. Cui Y, Tran S, Tinker A, Clapp LH. The molecular composition of K_ATP channels in human pulmonary artery smooth muscle cells and their modulation by growth. Am J Respir Cell Mol Biol 26: 135–143, 2002.[Abstract/Free Full Text]

8. Dai YP, Bungalon S, Hatton WJ, Hume JR, Yamboliev IA. ClC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery. Br J Pharmacol 145: 5–14, 2005.[CrossRef][Web of Science][Medline]

9. Flemming R, Cheong A, Dedman AM, Beech DJ. Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle. J Physiol (Lond) 543: 455–464, 2002.[Abstract/Free Full Text]

10. Frame MDS, Millanick MA. Mn and Cd transport by the Na-Ca exchanger of ferret blood cells. Am J Physiol Cell Physiol 261: C467–C475, 1991.[Abstract/Free Full Text]

11. Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J 12: 593–601, 1998.[Abstract/Free Full Text]

12. Golovina VA, Blaustein MP. Spatially and functionally distinct Ca²⁺ stores in sarcoplasmic and endoplasmic reticulum. Science 275: 1643–1648, 1997.[Abstract/Free Full Text]

13. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, Yuan JX. Upregulated TRP and enhanced capacitative Ca²⁺ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280: H746–H755, 2001.[Abstract/Free Full Text]

14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]

15. Hirota S, Pertens E, Janssen LJ. The reverse mode of the Na⁺/Ca²⁺ exchanger provides a source of Ca²⁺ for store refilling following agonist-induced Ca²⁺ mobilization. Am J Physiol Lung Cell Mol Physiol 292: L438–L447, 2007.[Abstract/Free Full Text]

16. Ihara E, Hirano K, Hirano M, Nishimura J, Nawata H, Kanaide H. Mechanism of down-regulation of L-type Ca²⁺ channel in proliferating smooth muscle cells of rat aorta. J Cell Biochem 87: 242–251, 2002.[CrossRef][Web of Science][Medline]

17. Janiak R, Wilson SM, Montague S, Hume JR. Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 280: C22–C33, 2001.[Abstract/Free Full Text]

18. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano K, Harada K, Miyamoto S, Nakazawa H, Won KJ, Sato K. Calcium movements, distribution and functions in smooth muscle. Pharmacol Rev 49: 157–230, 1997.[Abstract/Free Full Text]

19. Kuga T, Kobayashi S, HirakawaY, Kanaide H, Takeshita A. Cell cycle-dependent expression of L- and T-type Ca²⁺currents in rat aortic smooth muscle cells in primary culture. Circ Res 79: 14–19, 1996.[Abstract/Free Full Text]

20. Masuo M, Toyo-oka T, Shin WS, Sugimoto T. Growth-dependent alterations of intracellular Ca²⁺-handling mechanisms of vascular smooth muscle cells. PDGF negatively regulates functional expression of voltage-dependent, IP₃-mediated, and Ca²⁺-induced Ca²⁺ release channels. Circ Res 69: 1327–1339, 1991.[Abstract/Free Full Text]

21. McDaniel SS, Platoshyn O, Wang J, Yu Y, Sweeney M, Krick S, Rubin LJ, Yuan JX. Capacitative Ca²⁺ entry in agonist-induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 280: L870–L880, 2001.[Abstract/Free Full Text]

22. Ng LC, Gurney AM. Store-operated channels mediate Ca²⁺ influx and contraction in rat pulmonary artery. Circ Res 89: 923–929, 2001.[Abstract/Free Full Text]

23. Ng LC, Wilson SM, Hume JR. Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca²⁺ entry in isolated canine pulmonary arterial smooth muscle cells. J Physiol (Lond) 563: 409–419, 2005.[Abstract/Free Full Text]

24. Niggli E, Lederer WJ. Molecular operations of the sodium-calcium exchanger revealed by conformation currents. Nature 349: 621–624, 1991.[CrossRef][Medline]

25. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75: 487–517, 1995.[Abstract/Free Full Text]

26. Papp B, Enyedi A, Pászty K, Kovács T, Sarkady B, Gárdos G, Magnier C, Wuytack F, Enouf J. Simultaneous presence of two distinct endoplasmic-reticulum-type calcium- pump isoforms in human cells. Biochem J 288: 297–302, 1992.[Web of Science][Medline]

27. Parekh AB, Putney JW. Store-operated calcium channels. Physiol Rev 85: 757–810, 2005.[Abstract/Free Full Text]

28. Pittner J, Rhinehart K, Pallone TL. Ouabain modulation of endothelial calcium signaling in descending vasa recta. Am J Physiol Renal Physiol 291: F761–F769, 2006.[Abstract/Free Full Text]

29. Post JM, Gelband CH, Hume JR. [Ca²⁺]_i inhibition of K⁺ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization. Circ Res 77: 131–139, 1995.[Abstract/Free Full Text]

30. Robertson TP, Hague D, Aaronson PI, Ward JP. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J Physiol (Lond) 525: 669–680, 2000.[Abstract/Free Full Text]

31. Schwartz SM, Campbell CR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res 58: 427–444, 1986.[Abstract/Free Full Text]

32. Shima H, Blaustein MP. Modulation of evoked contractions in rat arteries by ryanodine, thapsigargin, and cyclopiazonic acid. Circ Res 70: 968–977, 1992.[Abstract/Free Full Text]

33. Smith JB, Cragoe EJ, Smith L. Na⁺/Ca⁺ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations. J Biol Chem 262: 11988–11994, 1987.[Abstract/Free Full Text]

34. Snetkov VA, Aaronson PI, Ward JP, Knock GA, Robertson TP. Capacitative calcium entry as pulmonary specific vasoconstrictor mechanism in small arteries of the rat. Br J Pharmacol 140: 97–106, 2003.[CrossRef][Web of Science][Medline]

35. Tanaka Y, Tashjian AH. Functional identification and quantitation of three intracellular Ca²⁺ pools in GH₄C₁ cells: evidence that the caffeine-responsive pool is coupled to a thapsigargin-resistant, ATP-dependent process. Biochemistry 32: 12062–12073, 1993.[CrossRef][Web of Science][Medline]

36. Tasker PN, Taylor CW, Nixon GF. Expression and distribution of InsP₃ receptor subtypes in proliferating vascular smooth muscle cells. Biochem Biophys Res Commun 273: 907–912, 2000.[CrossRef][Web of Science][Medline]

37. Trepakova ES, Gericke M, HirakawaY, Weisbrod RM, Cohen RA, Bolotina VM. The properties of a native cation channelactivated by Ca²⁺ store depletion in vascular smooth muscle cells. J Biol Chem 276: 7782–7790, 2001.[Abstract/Free Full Text]

38. Tribe RM, Borin ML, 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/Free Full Text]

39. Uehara A, Iwamoto T, Kita S, Shioya T, Yasukochi M, Nakamura Y, Imanaga I. Different cation sensitivities and binding site domains of Na⁺-Ca²⁺-K⁺ and Na⁺-Ca²⁺ exchangers. J Cell Physiol 203: 420–428, 2005.[CrossRef][Web of Science][Medline]

40. Vallot O, Combettes L, Jourdon P, Inamo J, Marty I, Claret M, Lompre AM. Intracellular Ca²⁺ handling in vascular smooth muscle cells is affected by proliferation. Arterioscler Thromb Vasc Biol 20: 1225–1235, 2000.[Abstract/Free Full Text]

41. Waldron RT, Short AD, Gill DL. Thapsigargin-resistant intracellular Ca²⁺ pumps. Role in calcium pool function and growth of thapsigargin-resistant cells. J Biol Chem 270: 11955–11961, 1995.[Abstract/Free Full Text]

42. Walker RL, Hume JR, Horowitz B. Differential expression and alternative splicing of TRP channel genes in smooth muscles. Am J Physiol Cell Physiol 280: C1184–C1192, 2001.[Abstract/Free Full Text]

43. Wang J, Shimoda LA, Sylvester JT. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 286: L848–L858, 2004.[Abstract/Free Full Text]

44. Wang J, Shimoda LA, Weigand L, Wang W, Sun D, Sylvester JT. Acute hypoxia increases intracellular [Ca²⁺] in pulmonary arterial smooth muscle by enhancing capacitative Ca²⁺ entry. Am J Physiol Lung Cell Mol Physiol 288: L1059–L1069, 2005.[Abstract/Free Full Text]

45. Wang Y, Chen J, Wang Y, Taylor CW, Hirata Y, Hagiwara H, Mikoshiba K, Toyo-oka T, Omata M, Sakaki Y. Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP₃) receptors in IP₃-induced Ca²⁺ release, capacitative Ca²⁺ entry, and proliferation of A7r5 vascualr smooth muscle cells. Circ Res 88: 202–209, 2001.[Abstract/Free Full Text]

46. Weigand L, Foxson J, Wang J, Shimoda LA, Sylvester JT. Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca²⁺ and nonselective cation channels. Am J Physiol Lung Cell Mol Physiol 289: L5–L13, 2005.[Abstract/Free Full Text]

47. Weirich J, Dumont L, Fleckenstein-Grun G. Contribution of capacitative and non-capacitative Ca²⁺-entry to M₃-receptor-mediated contraction of porcine coronary smooth muscle. Cell Calcium 38: 457–467, 2005.[CrossRef][Web of Science][Medline]

48. Wilson SM, Mason HS, Smith GD, Nicholson N, Johnston L, Janiak R, Hume JR. Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells. J Physiol (Lond) 543: 917–931, 2002.[Abstract/Free Full Text]

49. Zhang S, Yuan JX, Barrett KE, Dong H. Role of Na⁺/Ca²⁺ exchange in regulating cytosolic Ca²⁺ in cultured human pulmonary artery smooth muscle cells. Am J Physiol Cell Physiol 288: C245–C252, 2005.[Abstract/Free Full Text]

50. Zhang S, Dong H, Rubin LJ, Yuan JX. Upregulation of Na⁺/Ca²⁺ exchanger contributes to the enhanced Ca²⁺ entry in pulmonary artery SMC from patients with IPAH. Am J Physiol Cell Physiol 292: C2297–C2305, 2007.[Abstract/Free Full Text]

51. Zheng YM, Wang QS, Rathore R, Zhang WH, Mazurkiewicz JE, Sorrentino V, Singer HA, Kotlikoff MI, Wang YX. Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells. J Gen Physiol 125: 427–440, 2005.[Abstract/Free Full Text]

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