Divisions of 1Pulmonary and Critical Care Medicine and 2Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California
Submitted 20 August 2004 ; accepted in final form 23 September 2004
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ABSTRACT |
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sodium-calcium exchange; calcium homeostasis; vascular smooth muscle
Binding of vasoconstrictive and mitogenic agonists with G protein-coupled receptors (GPCR) and receptor tyrosine kinases in the plasma membrane activates phospholipase C, which causes hydrolysis of phosphatidylinositol and generation of cytosolic inositol 1,4,5-trisphosphate (IP3) and membrane-bound diacylglycerol. IP3 induces a rapid Ca2+ release through IP3 receptors at the SR membrane (14, 51), and the subsequent depletion of Ca2+ from the stores (i.e., the SR) opens a special family of Ca2+-permeable channels, namely, store-operated Ca2+ channels (SOC), and elicits capacitative Ca2+ entry (CCE) (5, 34, 37, 38). The store depletion-activated SOC are believed to be formed heterogeneously by different isoforms of transient receptor potential (TRP) channels (5, 11, 26, 32, 59). The homo- or heterotetrameric TRP channels are generally nonselective cation channels that allow both Ca2+ and Na+ to go through (29, 50, 64). Therefore, opening of TRP channels would increase both [Ca2+]cyt and cytosolic [Na+] as a result of Ca2+ and Na+ influx via the channels.
Mammalian cells maintain a low cytoplasmic concentration of Na+ ([Na+]cyt, 10 mM) compared with the extracellular concentration of Na+ ([Na+]out,
140 mM) because of the activity of the Na+-K+-ATPase (25, 58). The transmembrane Na+ gradient can be utilized to energize the Na+/Ca2+ exchanger, which moves Na+ and Ca2+ across the membrane in the opposite direction. Two families of plasma membrane Na+/Ca2+ exchanger proteins have been described in mammalian tissues (8, 30), one in which Ca2+ movement depends only on Na+ (NCX family) and the other in which Ca2+ movement depends on Na+ and K+ (NCKX family). The stoichiometry of NCX is 3 Na+ per 1 Ca2+, whereas that for NCKX is 4 Na+ per 1 Ca2+ and 1 K+. Both NCX and NCKX can operate in either a forward (Ca2+ exit) or reverse (Ca2+ entry) mode, depending on the Na+ and Ca2+ (and K+) gradients and membrane potential (Em) (8, 14, 24). For a constant extracellular [Ca2+] (1.82 mM) and [Na+] (
140 mM), [Ca2+]cyt is a cubic function of [Na+]cyt and an exponential function of Em. Therefore, a small change in [Na+]cyt or Em can cause a large change in [Ca2+]cyt (6).
Many functional studies have demonstrated that the plasma membrane Na+/Ca2+ exchanger is involved in regulating Ca2+ homeostasis of blood vessels (1, 8, 44, 45). The functional evidence for the sarcolemmal Na+/Ca2+ exchanger in vessels is also supported by direct demonstration that the Na+/Ca2+ exchanger is expressed in vascular smooth muscle and endothelial cells (23). However, the expression and function of the Na+/Ca2+ exchanger in human PASMC are unknown, although studies show that Na+/Ca2+ exchange activity is involved in the development of hypoxic pulmonary vasoconstriction in animals (8, 12, 27, 42, 57). Recently, it was also suggested that there is a functional and physical interaction of TRP cation channels with NCX proteins (40). Therefore, the purpose of this study was to confirm the functional expression of NCX and NCKX proteins and to characterize the role of the Na+/Ca2+ exchanger in store depletion-mediated Ca2+ entry in human PASMC, as well as the potential role of the Na+/Ca2+ exchange-driven Ca2+ entry in PASMC proliferation.
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METHODS AND MATERIALS |
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Western blot analysis. Human PASMC were gently washed twice in cold PBS, scraped into 0.3 ml of radioimmunoprecipitation assay buffer [1x PBS, 1% Nonidet P-40 (Amaresco, Solon, OH), 0.5% sodium deoxycholate, and 0.1% SDS], and incubated on ice for a 45-min period, during which the cell mixture was shaken for 30 s by vortex three times. Rat brain and pulmonary artery were stored at 80°C and then homogenized in a glass tube with a Teflon Dounce pestle in 5 ml of ice-cold assay buffer as described above. The resulting cell and tissue lysates were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were collected, and protein concentrations were determined using Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL) with BSA as a standard. Protein (30 µg) was mixed and boiled in 2x sample buffer (0.25 M Tris·HCl, pH 6.8, 20% glycerol, 8% SDS, and 0.02% bromphenol blue). Protein suspensions were electrophoretically separated on an 8% acrylamide gel, and protein bands were transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot cell transfer apparatus (Bio-Rad, Hercules, CA) under conditions recommended by the manufacturer. After 1 h of incubation in a blocking buffer (0.1% Tween 20 and 5% nonfat dry milk powder), the membranes were incubated with R3F1 monoclonal antibody against NCX1 (Swant, Bellinzona, Switzerland) diluted in blocking buffer (1:5,000) overnight at 4°C. Finally, the membranes were washed and exposed to anti-mouse horseradish peroxidase-conjugated IgG for 60 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).
RT-PCR. Total RNAs were extracted from human PASMC, rat brain, and rat pulmonary artery using the RNeasy mini kit (Qiagen, Valencia, CA). Genomic DNA was removed with RNase-free DNase according to the manufacturer's instructions. SuperScript reverse transcriptase (Invitrogen, Carlsbad, CA) was used to synthesize cDNA. RNA (2 µg) was first incubated with oligo(dT) (1 µl at 0.5 µg/µl) at 70°C for 10 min, and then 8 µl of a solution that contained 10x buffer, 10 mM dNTP, 20 mM MgCl2, 0.1 M DTT, 40 U/µl RNaseOUT, and 50 U/µl SuperScript II reverse transcriptase were added to the samples and incubated for 10 min at 30°C, 60 min at 42°C, and 5 min at 95°C. RNase-H (1 µl at 2 U/µl; GIBCO, Grand Island, NY) was added to each reaction. The sense and antisense primers were specifically designed from the coding regions of each gene (Table 1). The fidelity and specificity of the sense and antisense oligonucleotides were examined using the BLAST program.
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Measurement of [Ca2+]cyt. [Ca2+]cyt in single human PASMC was measured using the Ca2+-sensitive fluorescent indicator fura 2-AM. Cells on 25-mm coverslips were loaded with fura 2-AM (3 µM) for 30 min in the dark at room temperature (2224°C) under an atmosphere of 5% CO2-95% air. The fura 2-AM-loaded cells were then transferred to a perfusion chamber on the microscope stage and superfused with physiological salt solution (PSS) for 30 min to remove extracellular dye and allow intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. Fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illuminations) from the cells, as well as background fluorescence, was collected at room temperature (22°C) with the use of a x40 Nikon UV-Fluor objective and a charge-coupled device camera. The fluorescence signals emitted from the cells were monitored continuously using an Intracellular Imaging fluorescence microscopy system and were recorded in an IBM-compatible computer for later analysis. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 340 and 380 nm (F340/F380) using the ratio method based on the equation [Ca2+]cyt = Kd x (Sf2/Sb2) x (R Rmin)/(Rmax R), where Kd (225 nM) is the dissociation constant for Ca2+, R is the measured fluorescence ratio, and Rmin and Rmax are minimal and maximal ratios, respectively (20).
Cell cycle analysis. Human PASMC cell cycle distribution was analyzed using flow cytometry. Cells were first growth arrested in SmBM for 24 h and then cultured in 5% FBS-SmGM with or without KB-R7943 for 24 h. The cells were trypsinized, washed once with PBS, and fixed with 70% ethanol for at least 30 min at room temperature. The fixed cells were washed with PBS and incubated with a solution containing 50 µg/ml propidium iodine (Sigma) and 50 µg/ml RNase A (Sigma) for 30 min at room temperature in the dark. The stained cells were analyzed by FACS-Calibur with excitation at 488 nm and emission at 560640 nm (FL2 mode) using CellQuest software (Becton Dickinson, Mountain View, CA).
Measurement of cell number. Human PASMC were cultured in growth-arresting medium (SmBM) for 24 h and then switched to culture in 5% FBS-SmGM with or without KB-R7943 for 48 h. Cells were pelleted, washed twice with cold PBS, and resuspended in PBS for counting. Cell numbers were determined using a Z2 Coulter counter.
Chemicals. All chemicals were of analytical grade or better and were obtained from Fisher (Nepean, ON, Canada), BDH (Toronto, ON, Canada), or Sigma unless indicated otherwise. Pharmacological reagents were purchased from LC Laboratories (Woburn, MA), Seikagaku America (Falmouth, MA), or Calbiochem (San Diego, CA). Cell culture reagents were from Life Technologies (Rockville, MD).
Statistics and data analysis. Data are expressed as means ± SE; n represents the number of cells. Statistical analysis was performed using unpaired or paired Student's t-test or ANOVA as indicated. Differences were considered to be significant at P < 0.05.
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RESULTS |
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Functional role of Na+/Ca2+ exchanger in regulating [Ca2+]cyt. To test whether the Na+/Ca2+ exchanger is functionally involved in regulating [Ca2+]cyt, we first measured [Ca2+]cyt in human PASMC superfused with solutions with or without extracellular Na+. As shown in Fig. 2, removal of extracellular Na+ [by replacing Na+ in the bath solution with equimolar N-methyl-D-glucamine (NMDG+) or Li+] caused a rapid increase in [Ca2+]cyt as a result of switching the Na+/Ca2+ exchanger from the forward (Ca2+ exit) mode to the reverse (Ca2+ entry) mode (8, 28). Extracellular application of nifedipine (10 µM), a specific blocker of voltage-dependent Ca2+ channels, had no effect on the increase in [Ca2+]cyt driven by the reverse mode of the Na+/Ca2+ exchanger. However, extracellular application of KB-R7943, a potent and selective inhibitor of the Na+/Ca2+ exchanger (especially in its reverse mode), significantly attenuated the increase in [Ca2+]cyt induced by removal of extracellular Na+ (Fig. 2B). These results suggest that the Na+/Ca2+ exchanger can operate actively in the reverse mode in human PASMC.
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Involvement of Na+/Ca2+ exchanger in store depletion-induced Ca2+ entry. Upon activation of GPCR or receptor tyrosine kinases, IP3-mediated Ca2+ release (which induces an early transient increase in [Ca2+]cyt) depletes Ca2+ from intracellular stores (e.g., the SR). The store deletion-mediated opening of TRP channels would promote not only Ca2+ influx but also Na+ influx, because TRP channels are permeable to both Ca2+ and Na+ (35, 54). Because Ca2+ entry via the Na+/Ca2+ exchanger depends greatly on [Na+]cyt, the store depletion-mediated Na+ influx through TRP channels would increase [Na+]cyt, activate the reverse mode of Na+/Ca2+ exchange, and enhance Ca2+ entry. The next set of experiments was designed to test the hypothesis that in addition to triggering capacitative Ca2+ entry, the passive store depletion mediated by cyclopiazonic acid (CPA; 10 µM) induces Ca2+ entry as well via the Na+/Ca2+ exchanger.
In the absence of extracellular Ca2+ and presence of nifedipine (10 µM), extracellular application of CPA, a blocker of the Ca2+-Mg2+-ATPase in the SR (SERCA), induced a transient increase in [Ca2+]cyt due to Ca2+ leakage from the SR to the cytosol. Approximately 510 min later when the store was depleted (i.e., when the [Ca2+]cyt transients declined back to the basal level), restoration of extracellular [Ca2+] (to 1.8 mM) induced a large increase in [Ca2+]cyt due to Ca2+ entry (Fig. 3A). Extracellular application of KB-R7943 (10 µM; an inhibitor of the reverse mode of the Na+/Ca2+ exchanger) significantly attenuated the sustained phase of the store depletion-mediated Ca2+ entry (Fig. 3). Furthermore, removal of extracellular Na+ (by replacing external Na+ with equimolar NMDG+ or Li+) also markedly inhibited the store depletion-mediated Ca2+ entry (Fig. 4, A and C). In the absence of extracellular Na+ (i.e., when Na+/Ca2+ exchange was in the reverse mode or Ca2+ entry mode), the CPA-induced transient [Ca2+]cyt rise and baseline [Ca2+]cyt were both enhanced as a result of inhibited Ca2+ extrusion via the forward mode (or Ca2+ exit mode) of Na+/Ca2+ exchange (Fig. 4, A and B). These results indicate that the Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is involved in store depletion-mediated Ca2+ entry in human PASMC.
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DISCUSSION |
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The results from this study demonstrate that 1) human PASMC express NCX1 and NCKX3; 2) removal of extracellular Na+ activates the operation of the Na+/Ca2+ exchanger in the reverse mode to promote Ca2+ entry and increase [Ca2+]cyt; and 3) removal of extracellular K+ has no effect on the Ca2+ entry via the reverse mode of Na+/Ca2+ exchange. These observations provide strong evidence that Ca2+ entry via the reverse mode of the Na+/Ca2+ exchanger is a critical mechanism that regulates intracellular Ca2+ homeostasis in human PASMC. The NCX1 is probably the major Na+/Ca2+ exchanger isoform in human PASMC that functions in the reverse mode when [Na+]cyt is increased but works in the forward mode when [Ca2+]cyt is increased and [Na+]cyt remains unchanged. The protein expression level of NCX1 in cultured human PASMC was noticeably much smaller than that in freshly isolated rat pulmonary arteries (Fig. 1B), although the immunocytochemistry data clearly showed the surface expression of NCX1 in cultured PASMC (Fig. 1C). There are several possibilities that explain the discrepancies: 1) expression level of NCX1 may be significantly different between human and rat PASMC, and 2) expression level of NCX1 in human pulmonary arteries may be changed (e.g., downregulated) in cultured PASMC because of phenotypic changes (e.g., from contractile phenotype to synthetic or proliferative phenotype).
As mentioned earlier, the stoichiometry of the NCX-encoded Na+/Ca2+ exchanger proteins is 3 Na+ per 1 Ca2+. Thus [Ca2+]cyt determined by NCX is mainly related to [Na+]cyt by the following equation:
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where F is the Faraday constant, R is the gas constant, and T is absolute temperature. The equation indicates that for a constant [Ca2+]out and [Na+]out, [Ca2+]cyt is positively proportional to the third power of [Na+]cyt; that is, a small increase in [Na+]cyt can cause a large increase in [Ca2+]cyt due to Na+/Ca2+ exchange.
In PASMC, activation of GPCR (e.g., endothelin receptor) or receptor tyrosine kinases (e.g., platelet-derived growth factor receptor) often leads to an increased synthesis of IP3 that mediates Ca2+ mobilization from the SR to the cytosol by activating IP3 receptors (9, 15, 18, 36). The subsequent depletion of Ca2+ from the SR opens SOC in the plasma membrane and causes further Ca2+ influx to maintain a high level of [Ca2+]cyt during contraction or proliferation. The store depletion-mediated Ca2+ entry (SDCaE) has been demonstrated to result mainly from capacitative Ca2+ entry through Ca2+-permeable SOC (5, 56, 63). However, the canonical TRP (TRPC) channels that form functional SOC (5, 10, 33, 52, 55) are also permeable to other cations, including Na+; some of the TRPC channels are actually more permeable to Na+ than to Ca2+ (5, 13, 19). In other words, store depletion-mediated opening of TRPC channels would allow both Ca2+ and Na+ to enter the cell. The store depletion-mediated Na+ entry (SDNaE) would thus induce a local rise in [Na+]cyt, which activates the operation of Na+/Ca2+ exchangers in the reverse mode and subsequently increases [Ca2+]cyt (1, 2).
The results from this study demonstrate that 1) the CPA-induced passive depletion of Ca2+ from the intracellular stores (i.e., the SR) increases [Ca2+]cyt as a result of store depletion-mediated Ca2+ entry, and 2) removal of extracellular Na+ or extracellular application of KB-R7943 (an inhibitor of the Na+/Ca2+ exchanger) (8, 16, 21, 43) significantly attenuates the store depletion-mediated Ca2+ entry. These data suggest that the reverse mode of Na+/Ca2+ exchange participates in store depletion-mediated Ca2+ entry. In other words, the store depletion-mediated Ca2+ entry in human PASMC is composed of two components: 1) capacitative Ca2+ entry through Ca2+-permeable SOC (or TRPC) channels and 2) Ca2+ entry via the reverse model of the Na+/Ca2+ exchanger.
Store depletion-mediated Ca2+ entry is an important stimulus for mitogen-mediated PASMC proliferation (5, 9, 34, 39). Our previous studies demonstrated that functional blockade of SOC with La3+, Ni2+, or SKF-96365 as well as downregulation of TRPC channels with siRNA or antisense oligonucleotides all inhibited PASMC growth in the presence of serum and growth factors (19, 26, 48, 60). Consistent with these observations, we show in this study that inhibition of the reverse mode of Na+/Ca2+ exchange with KB-R7943, in addition to reducing the amplitude of store depletion-mediated Ca2+ entry, significantly inhibits PASMC proliferation. These results suggest that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is, at least in part, involved in inducing the rise in [Ca2+]cyt required for PASMC proliferation.
Increasing evidence suggests that there is a functional interaction between the Na+/Ca2+ exchanger and SOC (40) or TRPC channels that are activated by store depletion (34). Under physiological conditions, opening of these nonselective cation channels not only results in Na+ influx to the restricted plasma membrane-SR junctional space but also causes membrane depolarization as a result of inward cationic currents. Both the increase in [Na+]cyt and membrane depolarization drive the plasma membrane Na+/Ca2+ exchanger to its reversed mode of operation, thereby transporting more Ca2+ into cell (1, 7, 28). In human PASMC, our data support the contention that store depletion-mediated Ca2+ entry is caused by both capacitative Ca2+ entry and NCX-mediated Ca2+ entry, and the latter is also involved in serum- and growth factor-mediated PASMC proliferation. Whether TRPC isoforms, as well as which ones, are functionally or physically interacted with NCX proteins in human PASMC remains unclear.
Together, our results indicate that function of the Na+/Ca2+ exchanger plays an important role in regulating [Ca2+]cyt in human PASMC. Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is a critical pathway for increasing [Ca2+]cyt, inducing pulmonary vasoconstriction, and stimulating PASMC proliferation. In patients with pulmonary arterial hypertension, sustained pulmonary vasoconstriction and pulmonary vascular medial hypertrophy (due to excessive PASMC proliferation) are the major causes of increased pulmonary arterial pressure (41, 47). Therefore, better understanding the functional role of the Na+/Ca2+ exchanger in the pulmonary vasculature may lead to development of novel therapeutic approaches for treatment of pulmonary vascular diseases such as idiopathic pulmonary arterial hypertension.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Arnon A, Hamlyn JM, and Blaustein MP. Ouabain augments Ca2+ transients in arterial smooth muscle without raising cytosolic Na+. Am J Physiol Heart Circ Physiol 279: H679H691, 2000.
3. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 17: 491500, 1995.[ISI][Medline]
4. Berridge MJ. Inositol trisphosphate and calcium signaling. Nature 361: 315325, 1993.[CrossRef][ISI][Medline]
5. Birnbaumer L, Zhu X, Jiang M, Boulay G, Peyton M, Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, and Birnbaumer M. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc Natl Acad Sci USA 93: 1519515202, 1996.
6. Blaustein MP. Physiological effects of endogenous ouabain: control of intracellular Ca2+ stores and cell responsiveness. Am J Physiol Cell Physiol 264: C1367C1387, 1993.
7. Blaustein MP and Golovina VA. Structural complexity and functional diversity of endoplasmic reticulum Ca2+ stores. Trends Neurosci 24: 602608, 2001.[CrossRef][ISI][Medline]
8. Blaustein MP and Lederer WJ. Sodium/calcium exchange: Its physiological implications. Physiol Rev 79: 763854, 1999.
9. Bootman MD, Lipp P, and Berridge MJ. The organisation and functions of local Ca2+ signals. J Cell Sci 114: 22132222, 2001.[ISI][Medline]
10. Boulay G, Brown DM, Qin N, Jiang M, Dietrich A, Zhu MX, Chen Z, Birnbaumer M, Mikoshiba K, and Birnbaumer L. Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry. Proc Natl Acad Sci USA 96: 1495514960, 1999.
11. Brough GH, Wu S, Cloffi D, Moore TM, Li M, Dean N, and Stevens T. Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway. FASEB J 15: 17271738, 2001.
12. Chakraborti S, Mandal A, Das S, and Chakraborti T. Inhibition of Na+/Ca2+ exchanger by peroxynitrite in microsomes of pulmonary smooth muscle: role of matrix metalloproteinase-2. Biochim Biophys Acta 1671: 7078, 2004.[ISI][Medline]
13. Clapham DE, Runnels LW, and Strübing C. The TRP ion channel family. Nat Rev Neurosci 2: 387396, 2001.[CrossRef][ISI][Medline]
14. Davis MJ and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387423, 1999.
15. Doi S, Damron DS, Horibe M, and Murray PA. Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278: L118L130, 2000.
16. Dong H, Dunn J, and Lytton J. Electrophysiological studies of the cloned rat cardiac NCX1.1 in transfected HEK cells: a focus on the stoichiometry. Ann NY Acad Sci 976: 159165, 2002.
17. Frank JS, Chen F, Garfinkel A, Moore E, and Philipson KD. Immunolocalization of the Na+-Ca2+ exchanger in cardiac myocytes. Ann NY Acad Sci 779: 532533, 1996.[ISI][Medline]
18. Frey N, McKinsey TA, and Olson EN. Decoding calcium signals involved in cardiac growth and function. Nat Med 6: 12211227, 2000.[CrossRef][ISI][Medline]
19. Golovina VA, Platoshyn O, Bailey CL, Wang J, Limsuwan A, Sweeney M, Rubin LJ, and Yuan JXJ. Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation. Am J Physiol Heart Circ Physiol 280: H746H755, 2001.
20. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 34403450, 1985.[Abstract]
21. Iwamoto T, Kita S, Uehara A, Inoue Y, Taniguchi Y, Imanaga I, and Shigekawa M. Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Na+/Ca2+ exchanger. Mol Pharmacol 59: 524531, 2001.
22. Iwamoto T, Watano T, and Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1. J Biol Chem 271: 2239122397, 1996.
23. Juhaszova M, Ambesi A, Lindenmayer GE, Bloch RJ, and Blaustein MP. Na+-Ca2+ exchanger in arteries: identification by immunoblotting and immunofluorescence microscopy. Am J Physiol Cell Physiol 266: C234C242, 1994.
24. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano KI, Harada KI, Miyamoto S, Nakazawa H, Won KJ, and Sato K. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev 49: 157230, 1997.
25. Kim KJ, Cheek JM, and Crandall ED. Contribution of active Na+ and Cl fluxes to net ion transport by alveolar epithelium. Respir Physiol 85: 245256, 1991.[CrossRef][ISI][Medline]
26. Landsberg JW and Yuan JXJ. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci 19: 4752, 2004.
27. Lau CW, Chan YC, Yao X, Chan FL, Chen ZY, and Huang Y. Nickel inhibits urocortin-induced relaxation in the rat pulmonary artery. Eur J Pharmacol 488: 169172, 2004.[CrossRef][ISI][Medline]
28. Lee CH, Poburko D, Sahota P, Sandhu J, Ruehlmann DO, and van Breemen C. The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol 534: 641650, 2001.
29. Lintschinger B, Balzer-Geldsetzer M, Baskaran T, Graier WF, Romanin C, Zhu MX, and Groschner K. Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels. J Biol Chem 275: 2779927805, 2000.
30. Lytton J, Li XF, Dong H, and Kraev A. K+-dependent Na+/Ca2+ exchangers in the brain. Ann NY Acad Sci 976: 382393, 2002.
31. McDaniel SS, Platoshyn O, Wang J, Yu Y, Sweeney M, Krick S, Rubin LJ, and Yuan JXJ. Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction. Am J Physiol Lung Cell Mol Physiol 280: L870L880, 2001.
32. Ng LC and Gurney AM. Store-operated channels mediate Ca2+ influx and contraction in rat pulmonary artery. Circ Res 89: 923929, 2001.
33. Niemeyer BA, Suzuki E, Scott K, Jalink K, and Zuker CS. The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell 85: 651659, 1996.[ISI][Medline]
34. Parekh AB and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901930, 1997.
35. Park KS, Kim Y, Lee YH, Earm YE, and Ho WK. Mechanosensitive cation channels in arterial smooth muscle cells are activated by diacylglycerol and inhibited by phospholipase C inhibitor. Circ Res 93: 557564, 2003.
36. Pitt BR, Weng W, Steve AR, Blakely RD, Reynolds I, and Davies P. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am J Physiol Lung Cell Mol Physiol 266: L178L186, 1994.
37. Putney JWJ. A model for receptor-regulated calcium entry. Cell Calcium 7: 112, 1986.[ISI][Medline]
38. Putney JWJ. Pharmacology of capacitative calcium entry. Mol Interv 1: 8494, 2001.
39. Putney JWJ, Broad LM, Braun FJ, Lièvremont JP, and Bird GSJ. Mechanisms of capacitative calcium entry. J Cell Sci 114: 22232229, 2001.[ISI][Medline]
40. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, and Groschner K. Ca2+ signaling by TRPC3 involves Na+ entry and local coupling to the Na+/Ca2+ exchanger. J Biol Chem 279, 2004.
41. Rubin LJ. Primary pulmonary hypertension. N Engl J Med 336: 111117, 1997.
42. Salvaterra CG, Rubin LJ, Schaeffer J, and Blaustein MP. The influence of the transmembrane sodium gradient on the responses of pulmonary arteries to decreases in oxygen tension. Am Rev Respir Dis 139: 933939, 1989.[ISI][Medline]
43. Shigekawa M and Iwamoto T. Cardiac Na+-Ca2+ exchange: molecular and pharmacological aspects. Circ Res 88: 864876, 2001.
44. Shimizu H, Borin ML, and Blaustein MP. Use of La3+ to distinguish activity of the plasmalemmal Ca2+ pump from Na+/Ca2+ exchange in arterial myocytes. Cell Calcium 21: 3141, 1997.[ISI][Medline]
45. Slodzinski MK, Juhaszova M, and Blaustein MP. Antisense inhibition of Na+/Ca2+ exchange in primary cultured arterial myocytes. Am J Physiol Cell Physiol 269: C1340C1345, 1995.
46. Somlyo AP and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231236, 1994.[CrossRef][ISI][Medline]
47. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89144, 1997.[CrossRef][ISI][Medline]
48. Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, and Yuan JXJ. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 283: L144L155, 2002.
49. Teubl M, Groschner K, Kohlwein SD, Mayer B, and Schmidt K. Na+/Ca2+ exchange facilitates Ca2+-dependent activation of endothelial nitric-oxide synthase. J Biol Chem 274: 2952929535, 1999.
50. Thyagarajan B, Poteser M, Romanin C, Kahr H, Zhu MX, and Groschner K. Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels. J Biol Chem 276: 4814948158, 2001.
51. Tribe RM, Borin ML, and Blaustein MP. Functionally and spatially distinct Ca2+ stores are revealed in cultured vascular smooth muscle cells. Proc Natl Acad Sci USA 91: 59085912, 1994.[Abstract]
52. Vaca L, Sinkins WG, Hu Y, Kunze DL, and Schilling WP. Activation of recombinant trp by thapsigargin in Sf9 insect cells. Am J Physiol Cell Physiol 267: C1501C1505, 1994.
53. Vanden Abeele F, Shuba Y, Roudbaraki M, Lemonnier L, Vanoverberghe K, Mariot P, Skryma R, and Prevarskaya N. Store-operated Ca2+ channels in prostate cancer epithelial cells: function, regulation, and role in carcinogenesis. Cell Calcium 33: 357373, 2003.[CrossRef][ISI][Medline]
54. Van Eylen F, Kamagate A, and Herchuelz A. A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70 kDa NH2-terminal protein. Cell Calcium 30: 191198, 2001.[CrossRef][ISI][Medline]
55. Vannier B, Peyton M, Boulay G, Brown D, Qin N, Jiang M, Zhu X, and Birnbaumer L. Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel. Proc Natl Acad Sci USA 96: 20602064, 1999.
56. Venkatachalam K, van Rossum DB, Patterson RL, Ma HT, and Gill DL. The cellular and molecular basis of store-operated calcium entry. Nat Cell Biol 4: E263E272, 2002.[CrossRef][ISI][Medline]
57. Wang YX, Dhulipala PK, and Kotlikoff MI. Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells. FASEB J 14: 17311740, 2000.
58. Xiao AY, Wei L, Xia S, Rothman S, and Yu SP. Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons. J Neurosci 22: 13501362, 2002.
59. Xu SZ and Beech DJ. TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells. Circ Res 88: 8487, 2001.
60. Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, and Yuan JXJ. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 284: C316C330, 2003.
61. Yuan JXJ, Aldinger AM, Juhaszova M, Wang J, Conte JVJ, Gaine SP, Orens JB, and Rubin LJ. Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98: 14001406, 1998.
62. Yuan XJ, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Ionic currents in rat pulmonary and mesenteric arterial myocytes in primary culture and subculture. Am J Physiol Lung Cell Mol Physiol 264: L107L115, 1993.
63. Zhu X, Jiang M, Peyton M, Boulay G, Hurst R, Stefani E, and Birnbaumer L. trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell 85: 661671, 1996.[ISI][Medline]
64. Zitt C, Obukhov AG, Strübing C, Zobel A, Kalkbrenner F, Lückhoff A, and Schultz G. Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J Cell Biol 138: 13331341, 1997.