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CELLULAR AND MOLECULAR

Cyclic AMP-Dependent, Protein Kinase A-Independent Activation of Extracellular Signal-Regulated Kinase 1/2 Following Adenosine Receptor Stimulation in Human Umbilical Vein Endothelial Cells: Role of Exchange Protein Activated by cAMP 1 (Epac1)

Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio (Y.F.); and Department of Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, Idaho (M.E.O.)

Received January 16, 2007; accepted June 11, 2007.

	Abstract

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A critical process in angiogenesis is endothelial cell proliferation, which requires activation of extracellular signal-regulated kinase (ERK)1/2. This study analyzed the pathway responsible for adenosine-induced ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVEC). Characterization with adenosine receptor (AR) agonists and antagonists and the AR mRNA profile demonstrated that stimulation of the A_2BAR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A_2BAR-mediated ERK1/2 phosphorylation to 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 3-{1-[3-(amidinothio)propyl]-1H-in-dol-3-yl}-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro31-8220) indicated that protein kinase C stimulation is not required. The response did not involve transactivation of receptors for epidermal growth factor or vascular endothelial growth factor (VEGF). The A_2BAR-mediated response required functional G_s and was mimicked by forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A_2BAR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A_2BAR-mediated ERK1/2 activation may involve exchange protein activated by cAMP (Epac), a cAMP-activated guanine nucleotide exchange factor for Rap GTPases. Reverse Transcription-polymerase chain reaction analysis detected Epac1 but not Epac2 in HUVEC. 8-(p-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), an Epac activator, stimulated ERK1/2 phosphorylation. Overexpression of Epac1 enhanced A_2BAR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A_2BAR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A_2BAR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A_2BAR-mediated ERK1/2 phosphorylation. In summary, Epac1 activation in HUVEC results in ERK1/2 activation, and this protein, at least in part, mediates response to the physiologically relevant event of A_2BAR stimulation.

With regard to cAMP-dependent, PKA-independent responses, identification of Epac has begun to elucidate mechanisms by which such effects may occur. Epac1 and Epac2 are cAMP-activated guanine nucleotide exchange factors for RapGTPases (de Rooij et al., 1998; Kawasaki et al., 1998), and stimulation of Epac has been associated with responses such as cell adhesion, insulin secretion, regulation of endothelial cell permeability, and other responses (Holz et al., 2006). In some cell types, Rap activation of B-Raf promotes ERK1/2 activation, and the cell-specific expression of B-Raf may partly determine the qualitative effect of cAMP elevation on ERK1/2 activity (Dugan et al., 1999; Takahashi et al., 2004). Thus, it is plausible that an Epac Rap B-Raf pathway may participate in ERK1/2 activation. However, although certain studies have reported that Epac activation may result in ERK1/2 phosphorylation (Lin et al., 2003; Chen et al., 2004; Keiper et al., 2004), other studies described a positive coupling between Epac1 and RapGTPases that does not result in ERK1/2 activation (Enserink et al., 2002; Wang et al., 2006). Keiper et al. (2004) described a pathway in which Epac-Rap2B coupling did not directly result in ERK1/2 activation, but rather it required stimulation of additional signaling proteins such as phospholipase C- and H-Ras. Furthermore, Wang et al. (2006) found that the subcellular location of Epac-mediated Rap1 activation may dictate the ability of this pathway to stimulate ERK1/2.

With regard to physiological responses, the role of cAMP in regulation of cell proliferation has been most extensively studied in the context of activation of G_s-coupled GPCRs. Proliferation of vascular EC is a critical process in angiogenesis, the development of new blood vessels from the preexisting vasculature. One of the most extensively studied GPCR-mediated angiogenic responses has been that which occurs upon adenosine receptor activation (Adair, 2005). Due to metabolic regulation during hypoxia, the interstitial adenosine concentration rises to levels that activate endothelial ARs and promote cell proliferation and migration. These responses have been demonstrated in isolated EC obtained from several types of blood vessels (Meininger and Granger, 1990; Sexl et al., 1997; Grant et al., 2001; Dubey et al., 2002), and they are reflected in in vivo models of angiogenesis, including wound healing (Montesinos et al., 1997) and the retinopathy of prematurity (Mino et al., 2001). Specifically, the A_2AAR and A_2BAR which both couple to G_s activation are implicated in responses occurring in isolated EC (Sexl et al., 1997; Dubey et al., 2002) and the intact animal (Montesinos et al., 1997; Mino et al., 2001). Furthermore, stimulation of the G_s-coupled ₂AR promotes ERK1/2 phosphorylation in HUVEC (Sexl et al., 1997). Signaling cascades activated by GPCRs in EC that ultimately result in ERK1/2 stimulation have not been fully delineated. In certain EC, cAMP accumulation has been associated with cell proliferation (Meininger and Granger, 1990; Dubey et al., 2002). However, another study (Sexl et al., 1997) reported that cAMP did not induce ERK1/2 activation in HUVEC and that A_2AAR-mediated ERK1/2 stimulation occurred independently of G_s activation. In addition, a role for AR-mediated VEGF release in promoting ERK1/2 activation in EC has been described previously (Grant et al., 1999; Feoktistov et al., 2002).

The goal of the present study was to delineate the signal transduction cascade responsible for AR-mediated ERK1/2 activation in HUVEC. It was found that the A_2BAR uses a pathway that is cAMP-dependent but that does not require activation of PKA to produce this response. Furthermore, it was found that Epac1 activation results in ERK1/2 stimulation and that signaling via Epac1 plays a role in ERK1/2 activation mediated by the A_2BAR.

	Materials and Methods

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Cell Culture. All cells were maintained at 37°C in a 5% CO₂-humidified incubator. HUVEC, purchased from Cambrex Bio Science (Walkersville, MD) (previously Clonetics), were plated in culture dishes precoated with 0.1% gelatin, and cells were grown in endothelial cell growth medium 2 (EGM2) (Cambrex Bio Science) supplemented with 4% fetal bovine serum (FBS). HUVEC were studied from passage 4 to 9. HMEC-1 cells (Ades et al., 1992) were obtained from Dr. F. J. Candal at the Centers for Disease Control and Prevention (Atlanta, GA). HMEC-1 cells were maintained on gelatincoated flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 unit/ml penicillin G, and 100 µg/ml streptomycin sulfate. Before treatment, HUVEC and HMEC-1 cells were serum-starved overnight in DMEM supplemented with 1% bovine serum albumin unless otherwise stated. HEK293 and 293T cells were maintained in DMEM supplemented with 10% FBS and antibiotics.

ERK1/2 Western Blotting. HUVEC and HMEC-1 cells were incubated with receptor antagonists or signal transduction inhibitors for 30 min, followed by the addition of stimulants for 5 min unless otherwise stated. Lysates were prepared, protein concentration was determined (Bio-Rad protein assay; Bio-Rad, Hercules, CA), and equal amounts (typically 25 µg) of protein were analyzed by Western blotting. Nitrocellulose membranes were immunoblotted with rabbit polyclonal phospho-p44/42 mitogen-activated protein kinase antibody (Cell Signaling Technology Inc., Beverly, MA), and after stripping in 62.5 mM Tris, 2% SDS, and 0.1 M -mercaptoethanol, pH 6.8, at 50°C, blots were reprobed with mouse monoclonal ERK2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After chemiluminescent detection, signals on films were quantitated by densitometry, and the value obtained for phospho-ERK2 was normalized to that for total ERK2.

PCR Analysis of AR and Epac Expression in HUVEC. Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was generated with SuperScript preamplification system (Invitrogen) and amplified by PCR using the AR subtype-specific or Epac isoform-specific primers listed in Table 1. PCR products were analyzed on ethidium bromide-stained 1% agarose gels, and they were verified by sequencing.

To detect changes in Epac1 mRNA expression in siRNA-transfected HUVEC, real-time PCR was used. Total RNA and first-strand cDNA was prepared as described above. Real-time PCR was performed in triplicate with the human Epac1 TaqMan Gene Expression Assay (Hs00183449_ml; Applied Biosystems, Foster City, CA), and human glyceraldehyde-3-phosphate dehydrogenase TaqMan Gene Expression Assay was used as internal control. A protocol of 50°C x 2 min, 95°C x 10 min followed by 40 cycles of 95°C x 15 s and 60°C x 1 min was performed in an Applied Biosystem 7300 real-time PCR thermocycler. Results were analyzed using the comparative Ct method.

Retroviral Expression. pMT2-HA-Epac1 was kindly supplied by Dr. J. Bos (University Medical Centre Utrecht, Utrecht, The Netherlands), and this construct was used for the subcloning of HA-epitope-tagged human Epac1 into the NcoI and BamHI sites of the retroviral vector pMMP (kindly provided by Dr. D. Mukhopadhyay, Mayo Clinic, Rochester, MN). HA-tagged Epac1GEF, an Epac1 construct in which the GEF domain is deleted, was created by inserting a stop codon after the glutamate at position 614 of Epac1. H-RasN17 cDNA was kindly supplied by Dr. N. Ratner (University of Cincinnati, Cincinnati, OH), and this construct was used to create pMMP containing HA-epitope-tagged RasN17. To prepare retrovirus, the procedure described by Zeng et al. (2001) was used with minor modifications. In brief, 293T cells were seeded at 3 x 10⁶ cells/100-mm plate 24 h before transfection. Effectene Transfection Reagent (QIAGEN, Valencia, CA) was used according to the manufacturer's instructions and as described below. Two micrograms of retroviral construct (lacZ/pMMP, Epac1/pMMP, Epac1GEF/pMMP, or HA-RasN17), 1.5 µg of pMD.MLV gag.pol, and 0.5 µgof pMD.G were mixed in 300 µl of EC buffer. The latter two helper constructs were kindly provided by Dr. R. Mulligan (Harvard Medical School, Boston, MA). Thirty-two microliters of Enhancer reagent was added to the DNA mixture, and the whole mixture was then incubated at room temperature for 2 min. Subsequently, 30 µlof Effectene reagent was added, and incubation continued at room temperature for 5 min. The DNA mixture was added dropwise to 293T cells, and it was replaced with fresh growth media after 16 h. Media were collected 48 h after transfection and filtered. Twentyfour hours before infection, HUVEC were seeded at a density of 5 x 10⁴ cells/35-mm dish. Two milliliters of retrovirus solution and 2 ml of EGM2 were added to HUVEC with 10 µg/ml Polybrene (Sigma-Aldrich, St. Louis, MO). After 48 h, HUVEC were starved for 4 h in DMEM supplemented with 1% bovine serum albumin before analysis. Staining for -galactosidase expression (in situ -galactosidase staining kit; Stratagene, La Jolla, CA) in lacZ-infected cells indicated an expression efficiency of 80 to 95%.

siRNA Transfection of HUVEC. For experiments examining 5'-[N-ethylcarboxamido]-adenosine (NECA) and 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) as agonists, an Epac1-directed siRNA obtained from Ambion (Austin, TX) was used. For those studies examining responses to forskolin, an Epac1-directed siRNA identical to that used previously (Kooistra et al., 2005) was obtained from Dharmacon RNA Technologies (Lafayette, CO). A nontargeting siRNA was purchased from Ambion, and it was used as control in all experiments. HUVEC were seeded at a density of 2 x 10⁵ cells/well in six-well plates, and they were used for transfection 24 h later. HUVEC were transfected with Oligofectamine (Invitrogen) according to the manufacturer's instructions. In brief, siRNA (10.5 µlof20 µM stock) and Oligofectamine (10 µl) were diluted, respectively, with 193.75 and 27.5 µl of Opti-MEM (Invitrogen) in separate tubes and incubated for 10 min at room temperature. The contents of the tubes were combined, mixed gently, and incubated for 15 min at room temperature. During this time, HUVEC cell culture medium was replaced with 0.8 ml of Opti-MEM. The siRNA transfection mix was then added to the cells, and after 4 h, EGM2 supplemented with an additional 10% FBS and lacking antibiotics was added to the cells. After 48 h, media were removed from the cells, and HUVEC were serum-starved overnight before analysis.

Rap1 Pull-Down Assay. After serum starvation, HUVEC were treated with vehicle, 10 µM NECA, or 5 µM forskolin for 2 min. Cells were rinsed twice with ice-cold phosphate-buffered saline, and then they were lysed in 500 µl of ice-cold Rap pull-down buffer consisting of 50 mM Tris, pH 7.4, 250 mM NaCl, 10 mM MgCl₂, 1% Nonidet P-40, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were clarified by centrifugation at maximal speed in an Eppendorf centrifuge. Protein concentration of the supernatant was determined, and equal amounts (1 mg) of protein were incubated with 20 µg of RalGDS-RBD agarose beads (Upstate Biotechnology, Lake Placid, NY) for 45 min at 4°C. The agarose beads were pelleted by brief centrifugation, washed three times with Rap pull-down buffer, and eluted by boiling for 5 min in Laemmli sample buffer containing 62.5 mM dithiothreitol. The eluate was loaded onto 13% polyacrylamide gels, and immunoblotting was performed with a Rap1 antibody (Santa Cruz Biotechnology, Inc.).

Data Analysis. All experiments were performed a minimum of three times. Results are expressed as mean ± S.E.M. Statistical difference between two groups of observations was tested by the paired Student's t test and that among three or more groups by one-way analysis of variance followed by a Newman-Keuls post test. p < 0.05 was considered significant.

Reagents. NECA, CGS 21680, MRS1754, 8Br-cAMP, and isoproterenol were obtained from Sigma-Aldrich. N6-cyclopentyladenosine (CPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), xanthine amine congener (XAC), and N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were obtained from Sigma/RBI (Natick, MA). ZM241385 was from Tocris Cookson Inc. (Ellisville, MO). SCH 58261 was kindly supplied by Schering Plough (Kenilworth, NJ). Forskolin, PKI, H89, Ro20-1724, GF109203X, and Ro31-8220 were obtained from Calbiochem (San Diego, CA). VEGF and epidermal growth factor (EGF) were from R&D Systems (Minneapolis, MN) and Invitrogen, respectively. 8CPT-2Me-cAMP was from Biolog Life Science Institute (Bremen, Germany). Anti-HA mouse monoclonal antibody (clone 12CA5) was from Roche Diagnostics (Indianapolis, IN), and the antibody for G_s was kindly provided by Dr. Tom Gettys (Medical University of South Carolina, Charleston, SC).

	Results

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A_2BAR Stimulation Mediates ERK1/2 Phosphorylation in HUVEC. To begin to explore AR-mediated ERK1/2 phosphorylation in HUVEC, cells were treated with a panel of AR agonists. The nonspecific AR agonist NECA at the concentration of 10 µM produced a 5.9 ± 0.8-fold increase in the phosphorylation of ERK1/2, whereas 1 µM CGS 21680, 1 µM CPA, and 1 µM IB-MECA (selective for A_2AAR, A₁AR, and A₃AR, respectively) did not stimulate ERK1/2 phosphorylation (Fig. 1A). NECA stimulated ERK1/2 phosphorylation in a dose-dependent manner (Fig. 1B). Time course experiments indicated that the NECA-induced ERK1/2 phosphorylation peaked at 5 to 10 min, and then it returned to the basal level by 1 h (data not shown). To explore signaling mechanisms, the identity of the responsible AR subtype was more extensively examined. XAC (1 µM), a nonselective AR antagonist, blocked the NECA-induced ERK1/2 phosphorylation, whereas neither 50 nM SCH 58261, selective for A_2AAR, nor 1 µM DPCPX, selective for A₁AR, inhibited NECA-induced ERK1/2 phosphorylation (Fig. 1C). In a separate analysis, 50 nM SCH 58261 inhibited by 75% the ability of CGS 21680 to activate adenylyl cyclase in PC-12 cells (data not shown), which is a well characterized A_2AAR response. This agonist and antagonist pharmacological profile strongly suggests that the A_2BAR can mediate ERK1/2 phosphorylation in HUVEC. Therefore, we examined the sensitivity of the NECA-induced response to MRS1754, a recently described A_2BAR-selective antagonist (Kim et al., 2000). MRS1754 (200 nM) inhibited NECA-induced ERK1/2 phosphorylation by 76.7 ± 8.9% (Fig. 1D). Similar concentrations of MRS1754 have been used to differentiate A_2AAR and A_2BAR functional responses in macrophages (Kreckler et al., 2006). Because previous reports (Sexl et al., 1997; Feoktistov et al., 2002) have indicated the presence of the A_2AAR in HUVEC, additional analysis with CGS 21680 was performed. Time course experiments revealed no stimulation of ERK1/2 phosphorylation by 1 µM CGS 21680 at intervals ranging from 5 to 120 min (data not shown). Furthermore, CGS 21680 at concentrations ranging from 0.1 nM to 1 µM was ineffective at promoting ERK1/2 phosphorylation (data not shown); thus, no biphasic response had been overlooked. This pharmacological characterization, which used a functional readout, was supported by RT-PCR analysis of HUVEC RNA using AR subtype-specific primers, as only A_2BAR mRNA was detected (Fig. 1E). Identical RT-PCR results were obtained with a separate lot of HUVEC obtained from Cambrex Bio Science.

View larger version (37K): [in this window] [in a new window]   Fig. 1. A2BAR mediates ERK1/2 phosphorylation in HUVEC. A, representative Western blot demonstrating the level of ERK1/2 phosphorylation following 5-min treatment of HUVEC with the nonselective AR agonist NECA at 10 µM, the A2AAR-selective agonist CGS 21680 at 1 µM, the A1AR-selective agonist CPA at 1 µM, and the A3AR-selective agonist IB-MECA at 1 µM. Histogram summarizes multiple experiments (***, p < 0.001 versus basal). B, ERK1/2 phosphorylation was assessed after HUVEC were treated with increasing concentrations of NECA for 5 min (**, p < 0.01; ***, p < 0.001 versus basal). C, ERK1/2 phosphorylation by 5-min treatment with 10 µM NECA was assessed following 30-min pretreatment with AR antagonists XAC at 1 µM, SCH 58261 at 50 nM, and DPCPX at 1 µM(***, p < 0.001 versus basal; ###, p < 0.001 versus NECA alone). D, ERK1/2 phosphorylation by 5-min treatment with 10 µM NECA was assessed following 30-min pretreatment with 200 nM MRS1754. (***, p < 0.001 versus basal; ###, p < 0.001 versus NECA alone). E, HUVEC total RNA was isolated and amplified by RT-PCR with AR subtype-specific primers. AR cDNAs were used as positive controls, and H2O was used as negative control. PCR products were visualized on ethidium bromide-stained 1% agarose gels.

PKC Activation Is Not Involved in A_2BAR-Mediated ERK1/2 Phosphorylation. Multiple pathways for GPCR-induced ERK1/2 activation have been described previously (Luttrell, 2003). The A_2BAR has been reported to couple to G_q/11 in endothelial cells (Feoktistov et al., 2002) and in HEK293 cells (Gao et al., 1999). Because PKC isoforms may be activated following G_q/11 stimulation, it was determined whether PKC has a role in A_2BAR-mediated ERK1/2 phosphorylation. HUVEC were pretreated with vehicle or the PKC inhibitors GF109203X at 5 µM or Ro31-8220 at 5 µM for 30 min before addition of 10 µM NECA. NECA-induced ERK1/2 phosphorylation was not inhibited by GF109203X, and it was enhanced by Ro31-8220 (Fig. 2). ERK1/2 phosphorylation induced by phorbol 12-myristate 13-acetate (PMA), a PKC activator, was substantially reduced by both GF109203X and Ro31-8220 (Fig. 2), indicating the effectiveness of these inhibitors under the conditions used. Thus, PKC is apparently not responsible for NECA-induced ERK1/2 phosphorylation in HUVEC.

View larger version (25K): [in this window] [in a new window]   Fig. 2. PKC inhibitors do not block A2BAR-mediated ERK1/2 phosphorylation. HUVEC were treated with the PKC inhibitors GF109203X (GFX) at 5 µM and Ro31-8220 (Ro) at 5 µM for 30 min before 5-min treatment with 10 µM NECA (A) or 0.1 µM PMA (B). Shown is a representative Western blot for each agonist and a histogram that summarizes multiple experiments. (***, p < 0.001 for treatment versus basal; ###, p < 0.001 for agonist in the presence versus absence of PKC inhibitor).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Receptor tyrosine kinase transactivation is not involved in A2BAR-mediated ERK1/2 phosphorylation. A, examination of EGF receptor transactivation. HUVEC were treated with the EGF inhibitor AG1478 at 50 nM for 30 min followed by a 5-min treatment with vehicle, 10 µM NECA, or 5 ng/ml EGF. Western blotting was performed to assess ERK1/2 phosphorylation. B, examination of VEGF receptor transactivation. HUVEC were treated for 30 min with 5 µM GF109203X (GFX) before addition of 10 µM NECA or 10 ng/ml VEGF for 5 min, and ERK1/2 phosphorylation was assessed by Western blot. Shown are representative Western blots with the histograms summarizing three experiments. *, p < 0.05 relative to cells treated with agonist in the absence of inhibitor.

A_2BAR-Mediated ERK1/2 Phosphorylation Occurs in a cAMP-Dependent, PKA-Independent Manner. The role of the G_s-adenylyl cyclase (AC) pathway in A_2BAR-mediated ERK1/2 phosphorylation was next analyzed. Forskolin (5 µM), an AC activator, and 10 mM 8Br-cAMP, a stable cell membrane-permeable cAMP analog, induced a 5.9 ± 0.3- and 5.5 ± 1.4-fold increase in ERK1/2 phosphorylation, respectively (Fig. 4A). Involvement of cAMP in the NECA-induced response was also supported by experiments performed with the phosphodiesterase inhibitor Ro 20-1724. HUVEC were pretreated with or without 20 µM Ro 20-1724 for 30 min followed by addition of 10 µM NECA or 10 ng/ml VEGF. Relative to control cells, the NECA-induced ERK1/2 phosphorylation in the presence of Ro20-1724 was increased by 2.0 ± 0.1-fold (p < 0.01), whereas the response to VEGF was not significantly different between control and Ro20-1724-treated cells. To assess the involvement of G_s, HUVEC were exposed to 200 ng/ml cholera toxin for 48 h to down-regulate G_s levels. Cholera toxin decreased the G_s protein level by 59 ± 12% relative to untreated cells (Fig. 4C). In control HUVEC, NECA induced a 6.7 ± 2.3-fold increase in ERK1/2 phosphorylation; however, this response was reduced to 2.6 ± 0.7-fold stimulation in cholera toxin-treated cells (Fig. 4B). Forskolin-induced ERK1/2 phosphorylation was not affected by cholera toxin. As noted, the NECA-induced ERK1/2 phosphorylation, although substantially reduced, was not abolished by cholera toxin. In that cholera toxin treatment decreased G_s to levels 40% of control, it is likely that this remaining G_s may allow residual NECA-induced ERK1/2 phosphorylation to be observed. However, attempts to further down-regulate G_s with increased cholera toxin concentrations or more prolonged exposure substantially decreased cell viability and precluded analysis.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Gs-AC-cAMP pathway is involved in the A2BAR-mediated ERK1/2 phosphorylation. A, HUVEC were treated for 5 min with vehicle, 5 µM forskolin (FOR), 10 mM 8Br-cAMP, or 10 µM NECA followed by Western blotting to assess ERK1/2 phosphorylation. Shown is a representative Western blot with a histogram summarizing multiple experiments (*, p < 0.05; **, p < 0.01 versus basal). B, HUVEC were treated with cholera toxin at 200 ng/ml for 48 h before addition of 10 µM NECA or 5 µM forskolin for 5 min, and ERK1/2 phosphorylation was assessed by Western blot. Shown is a representative Western blot with a histogram summarizing multiple experiments (*, p < 0.05; **, p < 0.01 versus basal; #, p < 0.05 for NECA in the presence versus absence of cholera toxin). C, Western blotting was used to assess Gs protein levels in the untreated (basal) samples in the absence (control) and presence of cholera toxin.

It was determined that a 5-min treatment of HUVEC with 10 µM NECA increased intracellular cAMP levels to 129 ± 0.5% of control, which is a response similar to that reported for A_2BAR activation in coronary EC (Meininger and Granger, 1990) and human microvascular EC (Nguyen et al., 2003). To further examine the role of adenylyl cyclase, putative inhibitors of the enzyme, dideoxyadenosine and MDL-12,330, were examined at various concentrations. Although both compounds blunted NECA-induced ERK1/2 phosphorylation, dideoxyadenosine and MDL-12,330 also inhibited response to PMA (data not shown), suggesting a nonselective effect of these compounds.

The role of PKA was examined next, as it is the classic immediate downstream effector of cAMP. Two chemically distinct PKA inhibitors, H89 at 10 µM and PKI at 5 µM, were used. As shown in Fig. 5, neither compound inhibited NECA- or forskolin-induced ERK1/2 phosphorylation in HUVEC. As a positive control, the sensitivity of the NECA- and forskolin-induced response to these inhibitors was examined in HEK293 cells, because it had been reported previously that forskolin-stimulated ERK1/2 phosphorylation in these cells was abolished by H89 (Gao et al., 1999). In HEK293 cells, H89 and PKI inhibited forskolin-induced ERK1/2 phosphorylation by 107 ± 3 and 71 ± 20%, respectively (Fig. 5). Likewise, the response to NECA was inhibited by H89 and PKI by 82 ± 3 and 59 ± 3%, respectively (Fig. 5). These findings demonstrate the efficacy of H89 and PKI in antagonizing the actions of PKA. Importantly, the results also demonstrate that in a cell type-dependent manner cAMP may promote PKA-mediated ERK1/2 activation. In sum, these data strongly indicate that A_2BAR-mediated ERK1/2 phosphorylation in HUVEC requires G_s stimulation and the resulting cAMP accumulation; yet, unlike other cell types such as HEK293 cells, it does not involve PKA activation.

View larger version (27K): [in this window] [in a new window]   Fig. 5. PKA is not involved in A2BAR-mediated ERK1/2 phosphorylation in HUVEC. HUVEC were treated for 30 min with 10 µM H89 (left) or 5 µM PKI (middle) followed by a 5-min treatment with 10 µM NECA or 5 µM forskolin (For). ERK1/2 phosphorylation was assessed by Western blotting. Identical parallel experiments were performed with HEK293 cells (right). Shown are representative Western blots with the histograms summarizing multiple experiments (*, p < 0.05; **, p < 0.01 versus basal; #, p < 0.05; ##, p < 0.01 for agonist in the presence versus absence of PKA inhibitor).

To extend the above-described findings in terms of both GPCR involvement and endothelial cell specificity, the ability of -adrenergic receptor activation to elicit ERK1/2 phosphorylation in HMEC-1 cells was examined. HMEC-1 is an immortalized cell line derived from human dermal microvascular endothelial cells (Ades et al., 1992). In HMEC-1 cells, 10 µM isoproterenol and 5 µM forskolin induced a 4.6 ± 0.6- and 4.1 ± 0.5-fold increase in ERK1/2 phosphorylation, respectively (Fig. 6). Response to these stimulants was unaffected by pretreatment with 5 µM PKI. In HMEC-1 treated with PKI alone, an 2-fold increase in ERK1/2 phosphorylation was observed; however, this value did not display statistical significance. This slight increase may suggest that in these endothelial cells that a basal level of PKA activity may inhibit ERK1/2 phosphorylation, which is in agreement with the negative effect of PKA on this response in other cell types (Stork and Schmitt, 2002; Dumaz and Marais, 2005).

View larger version (40K): [in this window] [in a new window]   Fig. 6. ERK1/2 phosphorylation in microvascular endothelial cells. HMEC-1 cells were treated without or with 5 µM PKI for 30 min before exposure to 10 µM isoproteronol (ISO) or 5 µM forskolin (FOR) for 10 min. ERK1/2 phosphorylation was then assessed. Shown is a representative Western blot with a histogram that summarizes three experiments (*, p < 0.05 versus basal).

View larger version (52K): [in this window] [in a new window]   Fig. 7. Epac expression and function in HUVEC. A, total RNA was isolated from HUVEC and HEK293 cells and was used to perform RT-PCR with Epac isoform-specific primers. H2O served as negative control. PCR products were visualized on ethidium bromide-stained agarose gels. B, HUVEC were treated with 100 µM 8CPT-2Me-cAMP for the time periods indicated, and ERK1/2 phosphorylation was assessed. Representative Western blot is shown with histogram summarizing multiple experiments (*, p < 0.05 versus basal).

To assess the ability of Epac1 stimulation to promote ERK1/2 phosphorylation in HUVEC, cells were treated with 8CPT-2Me-cAMP, a cAMP analog that specifically activates Epac but not PKA (Enserink et al., 2002). In HUVEC, 100 µM 8CPT-2Me-cAMP stimulated ERK1/2 phosphorylation by 2.0 ± 0.2-fold at 15 min of treatment, with a return to basal level by 30 min (Fig. 7B).

Role of Epac1 in cAMP-Mediated ERK1/2 Phosphorylation. To assess the functional involvement of Epac1 in cAMP-induced ERK1/2 phosphorylation, Epac1 was overexpressed in HUVEC via retroviral infection, and responses to NECA and forskolin were examined. lacZ-infected HUVEC were used as a control, and in these cells, NECA and forskolin induced a 14.6 ± 2.6- and 11.5 ± 1.3-fold increase in ERK1/2 phosphorylation, respectively (Fig. 8A). In Epac1-overexpressing cells, these responses were potentiated to 33.6 ± 3.3- and 34.9 ± 2.0-fold, respectively. In that it has been reported that Epac-mediated activation of the c-Jun NH₂-terminal kinases family of mitogen-activated protein kinase occurs independently of the guanine nucleotide exchange factor activity of Epac (Hochbaum et al., 2003), responses were examined in HUVEC that expressed a truncated Epac1 molecule in which the GEF domain was deleted (Epac1GEF). In these cells, ERK1/2 phosphorylation in response to NECA and forskolin was not significantly different from that observed in lacZ-infected HUVEC (Fig. 8A). Enhancement of ERK1/2 phosphorylation by Epac1 overexpression was specific for compounds that elicit an increase in intracellular cAMP, as the response to 10 ng/ml VEGF was similar following transduction of HUVEC with lacZ or Epac1 (Fig. 8B).

View larger version (40K): [in this window] [in a new window]   Fig. 8. Overexpression of Epac1 enhances A2BAR-mediated ERK1/2 phosphorylation. HUVEC were exposed to retrovirus encoding lacZ, wild-type Epac1 (WT-Epac1) or to a truncated Epac1 protein in which the GEF domain was deleted (Epac1GEF) for 48 h and then were starved for 4 h before 10-min treatment with agonists. A, overexpression of WT-Epac1 but not that of Epac1GEF augmented ERK1/2 phosphorylation induced by NECA and forskolin. Expression of both HA-tagged Epac1 constructs was detected by blotting with 12CA5 antibody. Shown is a representative Western blot and a histogram summarizing three independent experiments. All results are reported as -fold stimulation relative to lacZ-infected HUVEC in the basal condition. *, p < 0.001 versus that specific agonist in lacZ-infected HUVEC. B, effect of Epac1 overexpression is specific for NECA and forskolin, as ERK1/2 phosphorylation induced by 10 ng/ml VEGF is similar in HUVEC transduced with lacZ and Epac1. Blot is representative of three experiments with similar results.

Epac1-Directed siRNA Inhibits A_2BAR-Mediated and cAMP-Induced ERK1/2 Phosphorylation. Because no chemical inhibitors of Epac1 are currently available, an RNA interference approach was used to determine the necessity of Epac1 in A_2BAR-mediated ERK1/2 phosphorylation. In four experiments, transfection of HUVEC with Epac1-directed siRNA induced a 68.4 ± 8.9% inhibition of Epac1 mRNA expression relative to cells transfected with a control siRNA (Fig. 9A). As shown in Fig. 9B, in HUVEC transfected with negative control siRNA, NECA elicited a 6.8 ± 1.1-fold increase in ERK1/2 phosphorylation, and this response was reduced to 4.1 ± 0.8-fold by Epac1-directed siRNA. The 3.0 ± 0.6-fold increase in ERK1/2 phosphorylation induced by 8CPT-2Me-cAMP was abolished by Epac1-directed siRNA. In a separate set of experiments, the ability of forskolin to stimulate ERK1/2 phosphorylation in siRNA-transfected cells was examined. Forskolin-induced ERK1/2 phosphorylation in HUVEC transfected with control siRNA was 4.2 ± 0.8-fold, and it was reduced to 2.1 ± 0.5 in cells transfected with Epac1-directed siRNA.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Epac1-directed siRNA inhibits 8CPT-2Me-cAMP- and NECA-induced ERK1/2 phosphorylation. HUVEC were transfected with negative control siRNA or Epac1-directed siRNA. A, Epac1 expression was assessed by RT-PCR and is reported as the percentage of that in negative control siRNA-transfected cells (n = 4). (B). In parallel experiments, siRNA-transfected HUVEC were treated with 100 µM 8CPT-2Me-cAMP or 10 µM NECA for 10 min with ERK1/2 phosphorylation assessed by Western blotting. Shown is a representative Western blot with the histogram summarizing the four experiments. *, p < 0.05 versus NECA in HUVEC transfected with control siRNA. C, in a separate experiment, HUVEC were transfected with negative control siRNA or a distinct Epac1-directed siRNA. Cells were untreated (basal) or exposed to 5 µM forskolin (FOR) for 10 min, and ERK1/2 phosphorylation was assessed by Western blotting. *, p < 0.05 versus forskolin in HUVEC transfected with control siRNA.

View larger version (40K): [in this window] [in a new window]   Fig. 10. Expression of dominant-negative H-Ras does not affect NECA- or forskolin-induced ERK1/2 phosphorylation. HUVEC were exposed to retrovirus encoding lacZ or a hemagglutinin-tagged dominant-negative H-Ras construct (HA-RasN17) for 48 h and then were starved for 4 h before 10-min treatment with either 10 µM NECA, 5 µM forskolin (FOR), or 5 ng/ml EGF. Western blotting was used to assess ERK1/2 phosphorylation, and expression of HA-RasN17 was determined by blotting with 12CA5 antibody. Shown is a representative Western blot, and the histogram summarizes three independent experiments. *, p < 0.05 versus EGF in HUVEC transduced with lacZ.

View larger version (36K): [in this window] [in a new window]   Fig. 11. Rap1 expression and activity in HUVEC. A, Western blotting of HUVEC whole-cell lysates detected the expression of Rap1, and the 95-kDa isoform of B-Raf. B, Rap1 is activated following A2BAR stimulation. HUVEC were treated with vehicle, 10 µM NECA, or 5 µM forskolin for 2 min and then were lysed in Rap pull-down buffer. Equal amounts of total protein from each lysate were incubated with RalGDS-RBD agarose beads for 45 min at 4°C. Active Rap1 bound to agarose beads was isolated, resolved on SDS-polyacrylamide gel electrophoresis gels, and visualized with a Rap1 antibody. NECA and forskolin activated Rap1 by 2.2 ± 0.2- and 3.0 ± 0.1-fold, respectively (**, p < 0.01 versus basal). Inset, representative Western blot of pulled down (activated) Rap1 (first three lanes) and total Rap1 in 10 µg of total protein lysates (last three lanes).

View larger version (34K): [in this window] [in a new window]   Fig. 12. MEK inhibitor blocks A2BAR-mediated ERK1/2 phosphorylation. HUVEC were treated with 20 µM PD98059 (PD) for 30 min before 5-min treatment with 10 µM NECA or 5 µM forskolin (FOR). *, p < 0.05; ***, p < 0.001 versus basal; ##, p < 0.01 for agonists in the presence versus absence of PD98059.

	Discussion

Top Abstract Materials and Methods Results Discussion References

GPCRs engage several pathways to elicit ERK1/2 activation (Luttrell, 2003), with many of these pathways pertinent to the A_2BAR, as this receptor may couple to both G_s and G_q/11. Several present findings implicate G_s-AC-cAMP signaling in A_2BAR-mediated ERK1/2 phosphorylation in HUVEC. This conclusion is based upon results generated through the use of reagents that act as activators or inhibitors of multiple proteins that constitute this signal transduction cascade. Furthermore, the lack of effect of selective inhibitors indicates that activation of PKC or transactivation of receptor tyrosine kinases does not underlie A_2BAR-mediated ERK1/2 phosphorylation in HUVEC.

In that the A_2BAR-mediated ERK1/2 phosphorylation in HUVEC is associated with an increase in cAMP levels, yet it occurs in a PKA-independent manner, focus was placed on Epac as a candidate downstream signaling molecule. The qualitative effect of cAMP on ERK1/2 activation may reflect the cell type-dependent expression and activation of signaling molecules such as Epac, Rap1, and B-Raf (Dugan et al., 1999; Takahashi et al., 2004). This cascade is depicted in Fig. 13. Expression of Epac1, but not Epac2, in HUVEC was determined by RT-PCR, and Western blotting detected Rap1 and the 95-kDa isoform of B-Raf. Several findings support the functional role of Epac1 in ERK1/2 phosphorylation in HUVEC. The Epac activator 8CPT-2Me-cAMP stimulated ERK1/2 phosphorylation with a kinetic profile very similar to that of NECA and forskolin. Many studies analyzing Epac function have primarily relied on the ability of 8CPT-2Me-cAMP to elicit responses independently of PKA activation. Varying results have been obtained with regard to 8CPT-2Me-cAMP-induced ERK1/2 activation. In the initial characterization of this compound, Enserink et al. (2002) reported that 8CPT-2Me-cAMP, although activating Rap1, could not stimulate ERK1/2 phosphorylation in several cell lines, including Chinese hamster ovary, OVCAR3, PC-12, and HEK293T. Furthermore, Nishihara et al. (2004) observed that cAMP promoted ERK1/2 activation in T84 colon epithelial cells in a PKA-independent manner but that this response was not mimicked by 8CPT-2Me-cAMP. However, other studies have demonstrated that 8CPT-2Me-cAMP may promote ERK1/2 activation in HEK293 cells and N1E-115 neuroblastoma cells (Keiper et al., 2004) and in the MC4 osteoblast cell line (Chen et al., 2004). In PC-12 cells expressing the 5-hydroxytryptamine_7A receptor, stimulation of ERK1/2 by 8CPT-2Me-cAMP was only observed following treatment of cells with an adenylyl cyclase inhibitor (Lin et al., 2003). At present, the factors that may underlie this differential response to 8CPT-2Me-cAMP are not defined. It is possible that signaling proteins that couple Epac to ERK1/2 activation are expressed in a cell type-dependent manner. Furthermore, the findings of Wang et al. (2006) in AtT20 cells indicate that B-Raf expression and Epac-mediated Rap1 activation are distinctly localized subcellularly and that this compartmentalization does not permit productive coupling to ERK1/2.

View larger version (24K): [in this window] [in a new window]   Fig. 13. Schematic diagram of proposed model of A2BAR signaling to ERK1/2 phosphorylation. The A2BAR couples to Gs to promote activation of adenylyl cyclase and an increase in intracellular cAMP levels. cAMP activates EPAC, which may then activate a RapGTPase to promote stimulation of B-Raf. B-Raf may then ultimately promote ERK1/2 phosphorylation. Also shown is coupling of cAMP to PKA. In a cell type-dependent manner, PKA can either inhibit or activate ERK1/2 possibly through regulation of Raf-1.

A role for Epac1 in ERK1/2 activation is also supported by studies that have demonstrated enhanced agonist-induced ERK1/2 phosphorylation following Epac1 overexpression (Lin et al., 2003; Keiper et al., 2004) and as observed presently in HUVEC with a marked elevation of the NECA- and forskolin-stimulated response. This potentiation is specific for agents that elevate cAMP, and it requires guanine nucleotide exchange activity, as an Epac1 construct lacking the GEF domain did not mimic the effects of the full-length protein.

In the present study, perhaps the most direct evidence for a role of Epac1 in A_2BAR-mediated ERK1/2 phosphorylation is obtained from experiments in which siRNA was used to decrease Epac1 expression. As no chemical inhibitor of Epac is available, RNA interference has allowed the role of Epac1 to be determined in responses such as regulation of EC permeability (Kooistra et al., 2005) and induction of suppressor of cytokine signaling-3 (Sands et al., 2006). In HUVEC, the 70% reduction in Epac1 expression produced by siRNA resulted in an 50% decrease in NECA-induced ERK1/2 phosphorylation. Likewise, forskolin-induced ERK1/2 phosphorylation was reduced by 65%. Residual ERK1/2 phosphorylation may indicate that a low level of Epac1 permits the response to remain partially intact. However, in that 8CPT-2Me-cAMP-induced ERK1/2 phosphorylation was ablated by Epac1 siRNA, this possibility seems unlikely. Alternatively, A_2BAR-mediated and forskolin-stimulated ERK1/2 phosphorylation may use additional pathways. Mechanisms in addition to Epac1 stimulation may contribute to these responses, as ERK1/2 phosphorylation elicited by NECA and forskolin was typically at least 2-fold greater than that induced by 8CPT-2Me-cAMP. However, a role for the cAMP effector CNrasGEF is unlikely as disruption of signaling by Ras, the downstream target of this protein, does not affect response to NECA or forskolin.

Epac activation is associated with an increasing number of functional responses; however, no studies have reported a role for Epac in cell proliferation, which is of particular relevance in that the mitogenic effects of cAMP may partially or completely occur in a PKA-independent manner (Stork and Schmitt, 2002; Dumaz and Marais, 2005). We are currently exploring the involvement of Epac in the proliferative effects of adenosine and other mitogens in EC. It will also be of interest to assess the generality of Epac1 signaling to ERK1/2 activation and proliferation in EC other than HUVEC. EC of different vascular beds display marked heterogeneity in terms of structure and function, because EC vary in terms of their environmental signals and perhaps epigenetic characteristics (Aird, 2007). No reports have directly described a role for Epac1 in ERK1/2 activation in any type of EC. Our studies with HMEC-1 cells demonstrate cAMP elevation induces a PKA-independent ERK1/2 phosphorylation, and they suggest that involvement of Epac1 in this response extends to EC types in addition to HUVEC. Most examinations of Epac in other facets of EC biology have employed HUVEC. In terms of modulation of EC permeability, Epac1 seems to have similar roles in HUVEC and in brain microvascular EC (Wittchen et al., 2005), in pulmonary aortic EC (Cullere et al., 2005), and in an in vivo mouse model (Fukuhara et al., 2005).

In addition to G_s, the A_2BAR has been reported to couple to G_q/11 (Gao et al., 1999; Feoktistov et al., 2002); however, the present findings indicate that PKC activation does not underlie A_2BAR-mediated stimulation of ERK1/2 phosphorylation, as PKC inhibitors did not diminish the NECA-induced response. The responses observed in HUVEC are similar to those reported for the endogenous A_2BAR in HEK293 cells (Gao et al., 1999). Specifically, GF109203X was without effect, whereas NECA-induced ERK1/2 phosphorylation was augmented by Ro31-8220. Gao et al. (1999) suggested that this enhancement by Ro31-8220 may occur due to a greater activity of Ro31-8220 against PKC relative to GF109203X, and they postulated that PKC may attenuate cAMP-induced ERK1/2 activation. This model is consistent with responses in HUVEC.

The present findings obtained from both pharmacological analysis and RT-PCR indicate that activation of the A_2BAR results in ERK1/2 phosphorylation in HUVEC. The ability of the A_2AAR to mediate ERK1/2 phosphorylation was reported previously (Sexl et al., 1997) based on the ability of the A_2AAR-selective agonist CGS 21680 to elicit this response in HUVEC. In addition, a similar designation was made because adenosine-induced ERK1/2 phosphorylation in HUVEC was blocked by 100 nM ZM241385 (Wyatt et al., 2002). However, a K_i of 26 nM at the human A_2BAR for ZM241385 has been reported (Beukers et al., 2000). Based on the above-mentioned reports, we placed emphasis on detection of any response to the A_2AAR. CGS 21680, at multiple time points and concentrations, did not elicit ERK1/2 phosphorylation in HUVEC. In HMEC-1 cells, we observed substantial ERK1/2 phosphorylation by 1 µM CGS 21680 at 5 min of treatment (data not shown), which confirms our ability to detect response to this agonist. The A_2AAR-selective antagonist SCH 58261, used at the appropriate concentration, did not block NECA-induced ERK1/2 phosphorylation, although the response was sensitive to the MRS1754, which is A_2BAR-selective. The exclusive detection of A_2BAR mRNA by RT-PCR analysis not only supports the pharmacological characterization of ERK1/2 phosphorylation but also indicates that all NECA-mediated responses observed presently in HUVEC, e.g., adenylyl cyclase stimulation and Rap1 activation, are mediated by the A_2BAR. The discrepancies that exist between our present findings and those previously reported in regard to ERK1/2 activation are similar to the discrepancies reported by various laboratories in regard to both AR subtype mRNA and functional expression in HUVEC (Feoktistov et al., 2002; Liu et al., 2002; Wyatt et al., 2002). These differences probably exist due to donor heterogeneity in HUVEC collection as well as differences in culture conditions. Indeed, Moy et al. (2006) described marked differences in functional properties and second messenger activity in HUVEC that were maintained in culture media of varying formulations. Specifically, it was observed that substantial differences existed in the effect of elevated cAMP levels on cell permeability in HUVEC maintained in media recommended by Cambrex Bio Science (as used in the present study) or media less enriched in supplements. Thus, the presence of varying amounts of mitogens, growth factors, and other additives may have substantial phenotypic effects on HUVEC. Alterations in receptor expression with passage of primary HUVEC may also occur. In addition to ARs, other signaling proteins perhaps may be regulated by culture conditions. This may explain additional discrepancies, as it was reported that treatment of HUVEC with 8Br-cAMP did not induce ERK1/2 phosphorylation (Sexl et al., 1997), which is in contrast to the robust response observed with this compound and forskolin in the present study and as reported for forskolin (Sands et al., 2006).

In summary, Epac1 stimulation in HUVEC results in ERK1/2 activation. This Epac1-mediated pathway has a role in the ERK1/2 activation induced by A_2BAR signaling that is a physiologically relevant stimulus for angiogenesis. Further exploration of the role of Epac1 activation in EC proliferation is warranted.

	Acknowledgements

 
We are very grateful to Prof. J. Bos for the HA-Epac1 cDNA, to Dr. N. Ratner for the H-RasN17 cDNA, and Drs. D. Mukhopadhyay and R. Mulligan for the constructs necessary for retroviral expression. We also thank Dr. F. Candal for the HMEC-1 cell line. We appreciate the assistance of Dr. William Greenlee (Schering Plough) in obtaining SCH 58261 and Dr. Dennis McGraw (University of Cincinnati) for assistance with RT-PCR assays. The excellent technical contributions of Greg Motz and Amy Schuster are greatly appreciated.

	Footnotes

 
This work was supported in part by National Institutes of Health Grant CA79531-01.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.119933.

ABBREVIATIONS: ERK, extracellular signal-regulated kinase; PKA, protein kinase A; Epac, exchange protein activated by cAMP; GPCR, G protein-coupled receptor; EC, endothelial cell(s); AR, adenosine receptor; HUVEC, human umbilical vein endothelial cell(s); VEGF, vascular endothelial growth factor; EGM2, endothelial cell growth medium 2; FBS, fetal bovine serum; HMEC, human microvascular endothelial cell(s); DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; PCR, polymerase chain reaction; siRNA, small interfering RNA; HA, hemagglutinin; GEF, guanine nucleotide exchange factor; RGD, Arg-Gly-Asp-containing domain; 8CPT-2Me-cAMP, 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate; CGS 21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 8Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate; CPA, N⁶-cyclopentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; XAC, xanthine amine congener; IB-MECA, N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide; ZM241385, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol; SCH 58261, 5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine; PKI, protein kinase A inhibitor; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; Ro 31-8220, 3-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX); EGF, epidermal growth factor; RT-PCR, reverse transcription-polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; MDL-12,330, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride; AG-1478, 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline; AC, adenylyl cyclase.

¹ Current affiliation: Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Address correspondence to: Dr. Mark E. Olah, College of Pharmacy, Box 8334, Idaho State University, Pocatello, ID 83209. E-mail: olahm{at}otc.isu.edu

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