Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093-0725
Submitted 24 March 2004 ; accepted in final form 15 June 2004
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ABSTRACT |
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capacitative Ca2+ entry; proliferation; vascular smooth muscle
While increased pulmonary vasoconstriction can underlie the increased pulmonary vascular resistance (PVR) in patients with pulmonary arterial hypertension, pulmonary vascular remodeling due to increased proliferation of PASMC in the tunica media, resulting in medial hypertrophy, also contributes greatly to the increased PVR. Increased extracellular or interstitial ATP level has been found to be involved in the progression of pulmonary hypertension (38, 56) as well as in the regulation of cell cycle progression and cell proliferation (19, 36, 56), all of which depend on elevations in cytosolic free Ca2+ concentration ([Ca2+]i). Investigators at our laboratory previously showed that store-operated Ca2+ (SOC) influx through canonical transient receptor potential (TRPC) channels may underlie part of the agonist-mediated Ca2+ response via capacitative Ca2+ entry (CCE) in PASMC (21, 61, 72). In all of these studies, enhanced CCE influenced either vasoconstriction or proliferation. Along with TRPC1 and TRPC6, TRPC4 is a TRPC isoform that has been implicated in forming receptor-operated Ca2+ (ROC) and/or SOC channels in many cell types (2, 9, 17, 30, 39, 51, 60, 62, 66, 72).
In this study, we hypothesized that extracellular ATP induces human PASMC proliferation via upregulation of TRPC4 expression and increase in CCE. Furthermore, we proposed that ATP-induced phosphorylation of the cyclic AMP response element-binding protein (CREB), a critical transcription factor in the regulation of cell survival (14, 49, 64), is involved in the increased transcription of TRPC4 induced by ATP.
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MATERIALS AND METHODS |
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Rat PASMC were used in some experiments to compare the response to ATP with human PASMC. Rat PASMC were prepared from the left and right branches of the main pulmonary artery and the intrapulmonary arteries of male Sprague-Dawley rats (150200 g). Briefly, the isolated pulmonary arteries were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml collagenase (Worthington Biochemical). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscle was then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma) at 37°C. Approximately 4550 min later, PASMC were sedimented by centrifugation, resuspended in fresh medium, and placed onto petri dishes or coverslips. The cells were cultured in high-glucose (4.5 g/l) DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioFluids) and incubated in 5% CO2 at 37°C in a humidified atmosphere.
DNA synthesis determination and cell counting.
[3H]Thymidine incorporation was determined to evaluate DNA synthesis and cell proliferation. Briefly, human PASMC were seeded in 24-well microplates at a density of 2 x 104 cells/well, cultured in SMGM for 48 h, and growth arrested in SMBM for 24 h. Treated cells were then incubated in 0.2% FBS-SMGM with 1 µCi of [3H]thymidine added to the cells for at least 16 h. Incorporation of radioactivity into trichloroacetic acid-insoluble material was measured using a liquid scintillation counter. For ATP experiments, the growth-arrested cells were treated with 100 µM ATP for 24 h before [3H]thymidine incorporation was measured. For small interfering RNA (siRNA) experiments, cells were transfected with control siRNA with scrambled sequence (si-Cont) and TRPC4-siRNA sequences for 0, 24, 48, or 72 h in the continuous presence of 100 µM ATP before [3H]thymidine uptake measurements. The dose-response curve of [3H]thymidine uptake in response to ATP treatment was best fitted using the SigmaPlot program.
For cell-counting experiments, previously growth-arrested (with 24-h SMBM) human PASMC were cultured in 0.2% FBS-SMGM with ATP for 48 h. Cell viability after 48 h was determined using 0.45% trypan blue (Sigma). Cell number was determined using a hemocytometer. The cell counts in the four 1-mm2 corner squares of the hemocytometer were averaged to calculate the total number of PASMC (1 x 104/ml) in the cell suspension.
Western blot analysis.
Human PASMC were gently washed twice in cold phosphate-buffered saline (PBS), scraped into lysis buffer [1% Nonidet P-4 (Amaresco), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/l aprotinin], and incubated on ice for 30 min. Cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was discarded. In some experiments, cell lysates were treated with the peptide N-glycosidase F (20 U; New England Biolabs) overnight at 4°C. The protein concentration in the supernatant was determined using a bicinchoninic acid protein assay with bovine serum albumin serving as a standard. Ten- to 25-µl aliquots of protein were mixed and boiled in SDS-PAGE sample buffer for 5 min. The protein samples separated on 10% SDS-PAGE were then transferred to nitrocellulose membranes by electroblotting in a MINI Trans-Blot cell transfer apparatus (Bio-Rad Laboratories) according to the manufacturer's instruction. After incubation overnight at 4°C in a blocking buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with polyclonal antibodies against TRPC4 (Alomone Labs) and -actin (Sigma). Finally, the membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham). In some experiments, the protein kinase inhibitors N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and KT-5823 (KT) (Sigma) were incubated along with the cells for 48 h before protein extraction and Western blot analysis. Band intensity is reported in arbitrary units (a.u.), which are a measure of band intensity relative to
-actin controls. The time courses of ATP-mediated CREB phosphorylation and TRPC4 upregulation were best fitted using the SigmaPlot program.
Synthesis and transfection of siRNA.
The 21-nucleotide siRNA sequence targeting TRPC4 (sense: 5'-ACUCUUGGUUCAGAAAGGATT-3'; antisense: 5'-UCCUUUCUGAACCAAGAGUTT-3') and a scrambled TRPC4 sequence (sense: 5'-GUGUCGUAAGUUCAUCCGATT-3'; antisense: 5'-UCGGAUGAACUUACGACACTT-3') were synthesized, and these were purchased from Sequitur. The human PASMC grown in SMGM to 80% confluence were transfected with either siRNA (20 nM) using the Gene Porter 2 transfection reagent kit (Gene Therapy Systems). After transfection, cells were incubated at 37°C in serum-free culture medium (SMBM). Fifteen hours after transfection, fresh growth medium was added and the cells were left to recover at 37°C for 8 h before protein extraction. The efficiency of siRNA transfection was determined using fluorescence-labeled siRNA; fluorescence was visible only in siRNA-transfected cells.
Measurement of [Ca2+]i.
PASMC were loaded with the membrane-permeable acetoxymethyl ester form of fura 2 (fura 2-AM; 3 µM) for 30 min in the dark at room temperature (2224°C). The fura 2-AM-loaded cells were then superfused with a standard bath solution (see below for composition) for 20 min at 34°C to wash away extracellular dye and to permit intracellular cleavage of fura 2-AM to active fura 2 by esterases. The standard bath solution contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with 5 mol/l NaOH). Fura 2 fluorescence from the cells and background fluorescence were collected at 32°C using Nikon UV-Fluor objectives. The fluorescence signals emitted from the cells were monitored continuously using an intracellular imaging fluorescence microscopy system and recorded on a personal computer for later analysis. [Ca2+]i was calculated from fura 2 fluorescence emission excited at 340 and 380 nm (F340/F380) using the ratio method (21) based on the following equation:
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Transfection of cells with the recombinant adenovirus. A recombinant adenoviral vector containing a CREB mutant (AdCREBM1) in which the phosphorylation site at 133Ser was mutated to an alanine was kindly provided by Dr. Anthony J. Zeleznik (University of Pittsburgh, Pittsburgh, PA) (55). Human PASMC were transfected using the AdCREBM1 vector as described by Tokunou et al. (63). Briefly, confluent PASMC were washed twice with PBS and then incubated with AdCREBM1 (added [AdCREBM1] is 10-fold the number of cells in each culture dish) at room temperature in PBS. After 2 h of incubation, cells were washed three times with PBS and cultured in 0.5% FBS-SMBM for days before being used for experiments.
Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using unpaired Student's t-tests or ANOVA as indicated. P < 0.05 was considered significant.
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RESULTS |
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ATP upregulates protein expression of TRPC4 channels and enhances CCE in PASMC. Having previously established that TRPC channels and CCE are important in vascular smooth muscle cell proliferation (13, 61, 72), we first verified whether ATP could also affect CCE by regulating TRPC4 channel expression. Western blot analysis (Fig. 2A) showed that 100 µM ATP significantly increased TRPC4 expression over the span of 48 h, from 88 ± 2 a.u. at time 0 (just before ATP exposure) to 118 ± 1 and 136 ± 1 a.u. after 24 or 48 h of treatment, respectively (P < 0.001 vs. ATP 0 h). Because of its putative role in underlying SOC and/or ROC formation, enhanced TRPC4 protein expression may augment Ca2+ entry through SOC and/or ROC (13).
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We also considered the possibility that [Ca2+]i might be increased because of an alternate mechanism after CPA application in the presence of ATP and reintroduced Ca2+. More specifically, the combination of store depletion and loss of extracellular Ca2+ may elevate intracellular Na+ levels via TRPC4 and result in diminished Na+/Ca2+ exchange activity, as was suggested previously regarding TRPC3 channels in carbachol-stimulated human embryonic kidney (HEK-293) cells (53). Reintroduction of extracellular Ca2+ could cause the exchanger to operate in a reverse mode, thereby causing Ca2+ influx rather than its "normal" Ca2+ extrusion. ATP-treated human PASMC exposed to 10 µM KB-R9743, a Na+/Ca2+ exchange inhibitor, still exhibited a significant increase in both Ca2+ release [128 ± 7 nM control (n = 53), 246 ± 25 nM ATP (n = 66); P < 0.001] and CCE levels (145 ± 10 nM control, 291 ± 37 nM ATP; P < 0.001), with no change in resting Ca2+ levels (108 ± 4 nM control, 109 ± 7 nM ATP) (Fig. 2C). These data indicate that ATP-induced CCE is mediated primarily by SOC channels with little involvement of the reverse-mode Na+/Ca2+ exchanger.
Downregulation of TRPC4 mRNA using siRNA inhibits CCE and cell proliferation in PASMC. To confirm that the low-dose ATP-induced TRPC4 gene upregulation (Fig. 2A) is responsible for enhanced CCE (Fig. 2B), a critical question to address is whether TRPC4 is involved in CCE in human PASMC. The molecular identity of SOC responsible for CCE remains unclear. TRPC4 is a TRPC isoform that can form heterotetramers with TRPC1 and TRPC5 and is involved in forming native SOC in many cell types (39, 58). Therefore, we tested whether TRPC4 in human PASMC is involved in forming Ca2+ channels that are activated by passive store depletion using CPA.
Using a previously identified siRNA specifically targeting TRPC4 (si-TRPC4) (13) (Fig. 3A), we were able to inhibit ATP-mediated upregulation of TRPC4 protein expression by 77% (Fig. 3B). The control siRNA with scrambled sequence (si-Cont) had no effect on TRPC4 expression. Furthermore, in PASMC treated with 100 µM ATP for 48 h, inhibition of TRPC4 expression with si-TRPC4 markedly attenuated the CPA-mediated CCE (Fig. 3C), bringing the amplitude of CCE down to 137 ± 7 nM from a control (i.e., si-Cont) value of 447 ± 17 nM. These data suggest that TRPC4 is involved, at least in human PASMC, in forming heterotetrameric store depletion-activated SOC channels. In parallel, si-TRPC4 caused a significant decrease in ATP-induced PASMC proliferation (Fig. 3D) over a 72-h period compared with cells treated with si-Cont. These results suggest that ATP-mediated upregulation of TRPC4 protein expression contributes to the enhancement of CCE and proliferation in human PASMC.
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Upregulated TRPC4 expression by ATP involves protein kinase activation. As shown in Fig. 4A, the ATP-mediated increase in CREB phosphorylation was due mainly to activation of suramin-sensitive P2 receptors. At this stage, we cannot ascertain whether CREB phosphorylation occurs because of stimulation of P2x or P2y receptors. Suramin can block both P2x (42) receptors, which form ligand-gated channels, and P2y receptors (65), which belong to the superfamily of G protein-coupled receptors (GPCR) (7, 65). GPCR are coupled to many signaling pathways, including those involved in protein kinase-mediated phosphorylation. CREB phosphorylation at 133Ser by protein kinase A (PKA) in response to cAMP and other kinases is sufficient to induce the transcription and expression of several genes. PKA in particular acts as a central processing hub not only in mediating but also in transmitting the effects of cAMP to different effector proteins, such as PKC, PKB, and mitogen-activated protein kinase (MAPK) (7, 50). Protein kinases may therefore be involved in the signal transduction pathway that occurs between plasmalemmal receptor binding by ATP and CREB phosphorylation before TRPC4 gene transcription.
We verified that PKA and PKG might be involved in the enhanced TRPC4 transcription and expression induced by 100 µM ATP. Inhibition of PKA or PKG with 10 µM H-89 or 2 µM KT-5823 (48-h treatments) significantly attenuated ATP-induced TRPC4 upregulation (Fig. 5). Taken together, these results suggest that ATP-mediated TRPC4 upregulation involves PKA- or PKG-induced phosphorylation of CREB.
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DISCUSSION |
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Effect of ATP on [Ca2+]i and CREB phosphorylation. In human PASMC, acute application of low doses (0.11 mM) of ATP did not evoke transient increases in [Ca2+]i (Fig. 1), while high-dose ATP caused a large, transient increase in [Ca2+]i. The former observation contrasts with reports showing that ATP at concentrations as low as 100 µM can cause cyclical Ca2+ oscillations in rat PASMC (present study), human coronary artery SMC, murine colonic SMC, and vascular endothelial cells (28, 31) mainly because of Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive sarcoplasmic reticulum (SR) stores. Therefore, the difference in Ca2+ reactivity to low-dose ATP we observed may be attributed to the different expression levels of sarcolemmal purinoceptors and the different sensitivities of IP3 receptors in the SR membrane in human PASMC relative to the other preparations.
In spite of the inability to induce transient increases in [Ca2+]i, long-term treatment with the low dose of ATP markedly upregulated TRPC4 expression (Fig. 2A), enhanced CCE amplitude (Fig. 2B), and increased proliferation (Fig. 1C) in human PASMC. Furthermore, ATP-mediated CREB phosphorylation preceded the ATP-induced increases in TRPC4 expression (Fig. 4Bb). Inhibition of CREB phosphorylation by overexpressing a nonphosphorylatable CREB mutant in human PASMC abolished ATP-mediated upregulation of TRPC4 and significantly inhibited ATP-mediated cell proliferation (data not shown). Taken together, these results suggest that treatment of human PASMC with ATP, at concentrations insufficient to cause an acute increase in [Ca2+]i, can phosphorylate CREB, upregulate TRPC4 expression, enhance CCE, and stimulate proliferation (Fig. 6).
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Mechanisms involved in ATP-mediated CREB phosphorylation and TRPC4 upregulation. Thrombin-induced aortic SMC proliferation and hypertrophy are mediated by CREB-dependent gene transcription of the c-fos gene (63). Inhibition of CREB phosphorylation attenuates neointimal proliferation in rat carotid artery by downregulating Bcl-2, an anti-apoptotic protein (64). Therefore, CREB is a transcription factor that plays an important role in the vascular remodeling process.
Various kinases are able to phosphorylate CREB, including PKA (22), PKG, PKC (69), Ca2+/calmodulin-dependent protein kinases (CaM) (8, 59), Akt/protein kinase B (45), MAPK (3), and Ras-dependent protein kinase (52). The phosphorylation of CREB on 133Ser enables CREB to bind to the cAMP response element (CRE) and to modulate transcription of the genes (e.g., bcl-2, c-fos) whose promoters contain a CRE binding sequence. In the present study, we have demonstrated that ATP-induced TRPC4 upregulation was attenuated when PKA and PKG were inhibited (Fig. 5), indicating that CREB was activated or phosphorylated by PKA/PKG-dependent pathways when human PASMC were treated with low-dose ATP for 424 h. The initial PKA/PKG-mediated CREB phosphorylation appeared not to be dependent on transient increases in [Ca2+]i, because acute application of 100 µM ATP had little effect on [Ca2+]i in human PASMC (Fig. 1A).
The phosphorylated CREB, by binding to the CRE, can upregulate many proteins and factors that regulate cell proliferation and survival (3, 32, 48, 49, 63). Although our observations provide evidence that ATP-mediated upregulation of TRPC4 is related to phosphorylation of CREB, it is still unclear how CREB modulates TRPC4 gene transcription. In other words, we still do not know whether ATP-mediated TRPC4 upregulation is due directly to binding of CREB to CRE in the TRPC4 gene promoter or indirectly to a CREB-induced intermediate (e.g., c-Fos or c-Jun) that subsequently upregulates or facilitates TRPC4 gene transcription.
In contrast to our finding that CREB phosphorylation preceded TRPC4 upregulation in human PASMC treated chronically with ATP, a recent study (46) suggested that enhanced SOC entry (but not Ca2+ entry through L-type voltage-dependent Ca2+ channels) led to CREB phosphorylation and c-fos transcription in rat and human cerebral arteries and rat aorta. In light of these new findings, it is apparent that upregulation of TRPC4 can initially be caused by Ca2+-dependent and -independent mechanisms. The subsequent enhancement of CCE and rises in [Ca2+]i may further upregulate TRPC4 gene expression, forming a positive feedback mechanism to ensure the increased [Ca2+]i required for cell cycle progression and PASMC proliferation. These observations also suggest that the physiological role of TRPC4 in the regulation of vascular contractility and VSMC proliferation is complex.
Role of TRPC channels and CCE in PASMC proliferation. Voltage-independent SOC channels are sensitive to changes in SR Ca2+ content and are activated upon SR depletion. TRPC gene products have been suggested as potential underlying components of native SOC in the cardiovascular system (16, 39, 43). TRPC1, TRPC4, and TRPC6 figure prominently as components of native SOC in pulmonary VSMC and endothelial cells and are involved in regulating vascular tone, microvascular permeability, and cell proliferation (5, 13, 17, 21, 33, 40, 61, 62, 68, 70, 72).
In the present study, we have shown that ATP significantly upregulated TRPC4 gene expression (Fig. 2A). TRPC3 was upregulated only at high ATP concentrations, while TRPC6 expression was unaffected by ATP treatments (data not shown). In parallel with the increased expression of TRPC4, CPA-induced CCE was also upregulated in PASMC treated with ATP (Fig. 2B). Both the ATP-induced TRPC4 upregulation and CCE enhancement were abolished by TRPC4-targeted siRNA application (Fig. 3, B and C), as was ATP-induced proliferation (Fig. 3D). These observations strongly suggest that ATP-induced human PASMC proliferation is partially mediated by the upregulation of tetrameric SOC comprising part of TRPC4 proteins.
A rise in [Ca2+]i is essential for cell proliferation (1, 35); removal or chelation of extracellular Ca2+ significantly inhibits PASMC proliferation in the presence of serum and growth factors (21). In the cell cycle, there are at least four CaM-sensitive steps: 1) the G0-to-G1 transition, 2) the G1-to-S transition, 3) the G2-to-M transition, and 4) the M phase (1, 35). Although low-dose ATP does not trigger a transient rise in [Ca2+]i, the upregulated TRPC4 channels and enhanced CCE resulting from long-term ATP treatment would amplify and help to maintain mitogen-induced increases in [Ca2+]i, accelerate transitions in the cell cycle, and stimulate PASMC proliferation (Fig. 6). Maintenance of sufficient Ca2+ within the SR is also required for cell growth, and depletion of the SR Ca2+ store induces growth arrest (27, 54) and triggers apoptosis (26). A recent study by Rosker et al. (53) also suggested that Na+ influx via TRPC3 channels might influence Na+/Ca2+ exchanger activity in carbachol-treated HeLa cells, leading to enhanced Ca2+ influx via reverse-mode Na+/Ca2+ exchange upon reintroduction of extracellular Ca2+. Our data (Fig. 2C) clearly demonstrate that Na+/Ca2+ exchange plays no role in the ATP-mediated enhancement of CCE. Because CCE is critical in refilling SR Ca2+ stores and in maintaining [Ca2+]SR, increased CCE amplitude due to enhanced TRPC4 expression may also participate in promoting cell proliferation by increasing [Ca2+]SR in human PASMC.
Physiological and pathophysiological implications. In light of its mitogenic effects and its multiple sources, extracellular ATP may play a critical role in the development of pulmonary vascular disease (37, 38, 56). In hypoxia-mediated pulmonary hypertension, for example, extensive pulmonary vascular remodeling can occur in addition to increased vasoconstriction, with both elements contributing to increased PVR and PAP. While much of the medial hypertrophy is due to PASMC hyperplasia and/or hypertrophy (11, 57), hypoxia also triggers the differentiation of adventitial fibroblasts and medial quiescent PASMC to synthetic phenotypes, leading to increased cell growth (10, 56). More recently, compelling evidence has indicated that, during hypoxia, ATP released from endothelial cells and fibroblasts induces fibroblast transdifferentiation to myofibroblasts, DNA synthesis, and proliferation of pulmonary adventitial fibroblasts (19, 56, 57). Our observations suggest that, under normoxic conditions, low-level ATP stimulation is sufficient to cause PASMC proliferation via a CREB- and TRPC4-dependent pathway. Therefore, small increases in extracellular ATP concentration may play a significant role in PASMC proliferation. Our findings may be relevant in unraveling the pathophysiological basis of severe pulmonary 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. Birnbaumer L. TRPC4 knockout mice: the coming of age of TRP channels as gates of calcium entry responsible for cellular responses. Circ Res 91: 13, 2002.
3. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, and Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms. Science 286: 13581362, 1999.
4. Born GV and Kratzer MA. Source and concentration of extracellular adenosine triphosphate during haemostasis in rats, rabbits and man. J Physiol 354: 419429, 1984.[Abstract]
5. 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.
6. Burnstock G. Integration of factors controlling vascular tone. Anesthesiology 79: 13681380, 1993.[ISI][Medline]
7. Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22: 364373, 2002.
8. Cartin L, Lounsbury KM, and Nelson MT. Coupling of Ca2+ to CREB activation and gene expression in intact cerebral arteries from mouse: roles of ryanodine receptors and voltage-dependent Ca2+ channels. Circ Res 86: 760767, 2000.
9. Dalrymple A, Slater DM, Beech D, Poston L, and Tribe RM. Molecular identification and localization of Trp homologues, putative calcium channels, in pregnant human uterus. Mol Hum Reprod 8: 946951, 2002.
10. Das M, Bouchey DM, Moore MJ, Hopkins DC, Nemenoff RA, and Stenmark KR. Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 1563115640, 2001.
11. Durmowicz AG and Stenmark KR. Mechanisms of structural remodeling in chronic pulmonary hypertension. Pediatr Rev 20: e91e102, 1999.[Medline]
12. Eichinger MR and Walker BR. Segmental heterogeneity of NO-mediated pulmonary vasodilation in rats. Am J Physiol Heart Circ Physiol 267: H494H499, 1994.
13. Fantozzi I, Zhang S, Platoshyn O, Remillard CV, Cowling RT, and Yuan JX-J. Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 285: L1233L1245, 2003.
14. Finkbeiner S. CREB couples neurotrophin signals to survival messages. Neuron 25: 1114, 2000.[ISI][Medline]
15. Forrester T and Williams CA. Release of adenosine triphosphate from isolated adult heart cells in response to hypoxia. J Physiol 268: 371390, 1977.[ISI][Medline]
16. Freichel M, Schweig U, Stauffenberger S, Freise D, Schorb W, and Flockerzi V. Store-operated cation channels in the heart and cells of the cardiovascular system. Cell Physiol Biochem 9: 270283, 1999.[CrossRef][ISI][Medline]
17. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost C, Weißgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, and Nilius B. Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4/ mice. Nat Cell Biol 3: 121127, 2001.[CrossRef][ISI][Medline]
18. Gaba SJM, Bourgouin-Karaouni D, Dujols P, Michel FB, and Prefaut C. Effects of adenosine triphosphate on pulmonary circulation in chronic obstructive pulmonary disease. ATP: a pulmonary vasoregulator? Am Rev Respir Dis 134: 11401144, 1986.[ISI][Medline]
19. Gerasimovskaya EV, Ahmad S, White CW, Jones PL, Carpenter TC, and Stenmark KR. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-activated adventitial fibroblast growth: signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J Biol Chem 277: 4463844650, 2002.
20. Gitterman DP and Evans RJ. Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium induced calcium release. Br J Pharmacol 132: 12011208, 2001.
21. 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.
22. Gonzalez GA and Montminy MR. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59: 675680, 1989.[ISI][Medline]
23. Hardingham GE, Chawla S, Johnson CM, and Bading H. Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature 385: 260265, 1997.[CrossRef][ISI][Medline]
24. Hartley SA and Kozlowski RZ. Electrophysiological consequences of purinergic receptor stimulation in isolated rat pulmonary arterial myocytes. Circ Res 80: 170178, 1997.
25. Hassessian H and Burnstock G. Interacting roles of nitric oxide and ATP in the pulmonary circulation of the rat. Br J Pharmacol 114: 846850, 1995.[Abstract]
26. He H, Lam M, TS M, and Distelhorst CW. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol 138: 12191228, 1997.
27. Husain M, Bein K, Jiang L, Alper SL, Simons M, and Rosenberg RD. c-Myb-dependent cell cycle progression and Ca2+ storage in cultured vascular smooth muscle cells. Circ Res 80: 617626, 1997.
28. Hüser J and Blatter LA. Elementary events of agonist-induced Ca2+ release in vascular endothelial cells. Am J Physiol Cell Physiol 273: C1775C1782, 1997.
29. Ingerman CM, Smith JB, and Silver MJ. Direct measurement of platelet secretion in whole blood. Thromb Res 16: 335344, 1979.[ISI][Medline]
30. Jung S, Strotmann R, Schultz G, and Plant TD. TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells. Am J Physiol Cell Physiol 282: C347C359, 2002.
31. Kimura C, Oike M, and Ito Y. Hypoxia-induced alterations in Ca2+ mobilization in brain microvascular endothelial cells. Am J Physiol Heart Circ Physiol 279: H2310H2318, 2000.
32. Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, and Reusch JEB. cAMP response element-binding protein content is a molecular determinant of smooth muscle proliferation and migration. J Biol Chem 276: 4613246141, 2001.
33. Lee CH, Rahimian R, Szado T, Sandhu J, Poburko D, Behra T, Chan L, and van Breemen C. Sequential opening of IP3-sensitive Ca2+ channels and SOC during -adrenergic activation of rabbit vena cava. Am J Physiol Heart Circ Physiol 282: H1768H1777, 2002.
34. Lipp P, Thomas D, Berridge MJ, and Bootman MD. Nuclear calcium signalling by individual cytoplasmic calcium puffs. EMBO J 16: 71667173, 1997.
35. Lu KP and Means AR. Regulation of the cell cycle by calcium and calmodulin. Endocr Rev 14: 4058, 1993.[ISI][Medline]
36. Malam-Souley R, Campan M, Gadeau AP, and Desgrange C. Exogenous ATP induces a limited cell cycle progression of arterial smooth muscle cells. Am J Physiol Cell Physiol 264: C783C788, 1993.
37. McCormack DG, Barnes PJ, and Evans TW. Purinoceptors in the pulmonary circulation of the rat and their role in hypoxic vasoconstriction. Br J Pharmacol 98: 367372, 1989.[Abstract]
38. McCormack DG, Crawley DE, and Evans TW. New perspectives in the pulmonary circulation and hypoxic pulmonary vasoconstriction. Pulm Pharmacol 6: 97108, 1993.[CrossRef][ISI][Medline]
39. Minke B and Cook B. TRP channel proteins and signal transduction. Physiol Rev 82: 429472, 2002.
40. Ng LC and Gurney AM. Store-operated channels mediate Ca2+ influx and contraction in rat pulmonary artery. Circ Res 89: 923929, 2001.
41. Nishiyama A, Majid DSA, Taher KA, Miyatake A, and Navar LG. Relation between renal interstitial ATP concentrations and autoregulation-mediated changes in renal vascular resistance. Circ Res 86: 656662, 2000.
42. North RA and Surprenant A. Pharmacology of cloned P2x receptors. Annu Rev Pharmacol Toxicol 40: 563580, 2000.[CrossRef][ISI][Medline]
43. Parekh AB and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901930, 1997.
44. Pearson JD and Gordon JL. Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. Nature 281: 384386, 1979.[ISI][Medline]
45. Pugazhenthi S, Nesterova A, Sable C, Heidenreich KA, Boxer LM, Heasley LE, and Reusch JEB. Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem 275: 1076110766, 2000.
46. Pulver RA, Rose-Curtis P, Roe MW, Wellman GC, and Lounsbury KM. Store-operated Ca2+ entry activates the CREB transcription factor in vascular smooth muscle. Circ Res 94: 13511358, 2004.
47. Remillard CV, Zhang WM, Shimoda LA, and Sham JSK. Physiological properties and functions of Ca2+ sparks in rat intrapulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L433L444, 2002.
48. Reusch JEB and Klemm DJ. Cyclic AMP response element-binding protein in the vessel wall: good or bad? Circulation 108: 11641166, 2003.
49. Riccio A, Ahn S, Davenport CM, Blendy JA, and Ginty DD. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science 286: 23582361, 1999.
50. Robinson-White A and Stratakis CA. Protein kinase A signalling: "cross-talk" with other pathways in endocrine cells. Ann NY Acad Sci 968: 256270, 2002.
51. Rosado JA and Sage SO. The ERK cascade, a new pathway involved in the activation of store-mediated calcium entry in human platelets. Trends Cardiovasc Med 12: 229234, 2002.[CrossRef][ISI][Medline]
52. Rosen LB, Ginty DD, Weber MJ, and Greenberg ME. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron 12: 12071221, 1994.[ISI][Medline]
53. 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: 1369613704, 2004.
54. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, and Gill DL. Intracellular Ca2+ pool content is linked to control of cell growth. Proc Natl Acad Sci USA 90: 49864990, 1993.[Abstract]
55. Somers JP, DeLoia JA, and Zeleznik AJ. Adenovirus-directed expression of a nonphosphorylatable mutant of CREB (cAMP response element-binding protein) adversely affects the survival, but not the differentiation, of rat granulosa cells. Mol Endocrinol 13: 13641372, 1999.
56. Stenmark KR, Gerasimovskaya EV, Nemenoff RA, and Das M. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 122: 326S-334S, 2002.
57. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89144, 1997.[CrossRef][ISI][Medline]
58. Strübing C, Krapivinsky G, Krapivinsky L, and Clapham DE. TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29: 645655, 2001.[ISI][Medline]
59. Sun P, Lou L, and Maurer RA. Regulation of activating transcription factor-1 and the cAMP response element-binding protein by Ca2+/calmodulin-dependent protein kinases type I, II, and IV. J Biol Chem 271: 30663073, 1996.
60. Sweeney M, McDaniel SS, Platoshyn O, Zhang S, Yu Y, Lapp BR, Zhao Y, Thistlethwaite PA, and Yuan JX-J. Role of capacitative Ca2+ entry in bronchial contraction and remodeling. J Appl Physiol 92: 15941602, 2002.
61. Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, and Yuan JX-J. 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.
62. Tiruppathi C, Freichel M, Vogel SM, Paria BC, Mehta D, Flockerzi V, and Malik AB. Impairment of store-operated Ca2+ entry in TRPC4/ mice interferes with increases in lung microvascular permeability. Circ Res 91: 7076, 2002.
63. Tokunou T, Ichiki T, Takeda K, Funakoshi Y, Iino N, and Takeshita A. cAMP response element-binding protein mediates thrombin-induced proliferation of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21: 17641769, 2001.
64. Tokunou T, Shibata R, Kai H, Ichiki T, Morisaki T, Fukuyama K, Ono H, Iino N, Masuda S, Shimokawa H, Egashira K, Imaizumi T, and Takeshita A. Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells. Circulation 108: 12461252, 2003.
65. Von Kügelgen I and Wetter A. Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310323, 2000.[CrossRef][ISI][Medline]
66. Wang J, Laurier LG, Sims SM, and Preiksaitis HG. Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle. Am J Physiol Gastrointest Liver Physiol 284: G1074G1083, 2003.
67. Wellman GC, Cartin L, Eckman DM, Stevenson AS, Saundry CM, Lederer WJ, and Nelson MT. Membrane depolarization, elevated Ca2+ entry, and gene expression in cerebral arteries of hypertensive rats. Am J Physiol Heart Circ Physiol 281: H2556H2567, 2001.
68. Welsh DG, Morielli AD, Nelson MT, and Brayden JE. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res 90: 248250, 2002.
69. Xie H and Rothstein TL. Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig. J Immunol 154: 17171723, 1995.
70. 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.
71. Yamamoto K, Sokabe T, Ohura N, Nakatsuka H, Kamiya A, and Ando J. Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells. Am J Physiol Heart Circ Physiol 285: H793H803, 2003.
72. Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, and Yuan JX-J. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Am J Physiol Cell Physiol 284: C316C330, 2003.
73. Zhang WM, Yip KP, Lin MJ, Shimoda LA, Li WH, and Sham JSK. ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors. Am J Physiol Lung Cell Mol Physiol 285: L680L690, 2003.