(Hypertension. 2001;37:716.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
A2B Receptors Mediate the Antimitogenic Effects of Adenosine in Cardiac Fibroblasts
From the Center for Clinical Pharmacology, Departments of Medicine (R.K.D., D.G.G., Z.M., E.K.J.), and Pharmacology (L.C.Z., E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, and Clinic for Endocrinology (R.K.D.), Department of Obstetrics and Gynecology, University Hospital Zurich, Zurich, Switzerland.
Correspondence to Dr Raghvendra K. Dubey, Department of Obstetrics and Gynecology, Clinic for Endocrinology, D215, NORD-1; Frauenklinik, University Hospital Zurich, 8091 Zurich, Switzerland. E-mail raghvendra.dubey{at}fhk.usz.ch
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Abstract |
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Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis (3H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis (3H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N6-cyclopentyladenosine, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A2 receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A2B receptors coupled to inhibition of MAP kinase activity. Thus, A2B receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A2B receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.
Key Words: adenosine • cardiac fibroblast • A2B receptor • cardiac remodeling • left ventricular hypertrophy • myocardial infarction • proliferation
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Introduction |
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Abnormal growth of cardiac fibroblasts is importantly involved in the pathophysiology of cardiac remodeling induced by hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.1 Cardiac fibroblasts (CFs), which constitute 60% of the total heart cells, contribute to the pathologic structural changes in the heart by undergoing proliferation, deposition of extracellular matrix proteins such as collagen, and replacing myocytes with fibrotic scar tissue.2 Thus, CF-induced cardiac remodeling may participate in diastolic and systolic dysfunction, leading to congestive heart failure.1
CF growth is regulated by several autocrine/paracrine factors,1 including adenosine,3 which has long been known as a "retaliatory" metabolite, particularly in the heart, where it induces cardioprotective effects.3 The biological effects of adenosine are mediated by means of adenosine receptors, which exist in multiple subtypes (A1, A2A, A2B, and A3 receptors).3 Within the heart, the standard view is that mainly A1 and A2A adenosine receptors are cardioprotective. For example, activation of A1 receptors attenuates the sympathetic nervous system, inhibits renin release from juxtaglomerular cells and opens cardiac K+ channels.3 By means of activation of A2B receptors, adenosine causes vasodilation, inhibits platelet aggregation, diminishes neutrophil adhesion to vascular endothelial cells, attenuates neutrophil-induced endothelial cell damage, and stimulates nitric oxide release from vascular endothelial cells and vascular smooth muscle cells.4 5
Although the standard view is that A1 and A2A receptors are the most important with regard to adenosine-mediated cardioprotection, indirect evidence suggests that adenosine inhibits CF growth by means of activation of A2B receptors.6 7 However, due to lack of availability of selective A2B receptor antagonists or agonists, the inference that A2B receptors mediate the growth inhibiting effects of adenosine is inconclusive. Accordingly, in the present study, we developed antisense oligonucleotides against rat A2B receptors and used them to test the role of A2B receptors in regulating CF growth and MAP kinase activity.
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Methods |
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All tissue culture ware and reagents were purchased from GIBCO Laboratories. Fetal calf serum (FCS) was obtained from HyClone Laboratories Inc. Adenosine, 2-chloroadenosine (Cl-Ad), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and PDGF-BB were purchased from Sigma Chemical Co. N6-cyclopentyladenosine (CPA), CGS21680, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), iodotubericidin (IDO), 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX), 5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-methylcarboxamidoadenosine (MECA), and 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) were purchased from Research Biochemicals International. KF17837 was a gift from Kyowa Hakko Kogyo Co. Ltd, (Sunto, Shizuoka, Japan). 3H-Thymidine (specific activity, 11.8 Ci/mmol) and L-3H-proline (23 Ci/mmol) were purchased from NEN. All other reagents were of tissue culture or best grade available.
Cardiac Fibroblast Cultures
Hearts were surgically removed from
anesthetized (50 mg/kg intraperitoneal
injection of pentobarbital) Sprague-Dawley male rats (150 to 200
g; n=14), and left ventricular CFs cultured using enzymatic
digestion with collagenase and selective plating as
described by us previously.6
CF purity of >98% was confirmed by morphology (CFs are thin,
triangular cells with light cytoplasm) and
immunostaining (negative immunostaining
against sarcomeric actin, desmin, von Willebrand factor VIII,
and positive immunostaining with
antivimentin).6 CFs in first
or second passage were used for all experiments.
Antisense Oligonucleotides for
A2B Receptors
We used the GenBank database to obtain the rat
A2B receptor cDNA sequence and used MacVector
4.1 to select an antisense oligonucleotide with
complementary sequences to the A2B receptor mRNA
(5'-CTCGTGTTCCAGTGACCAA-3'). Midland Certified Reagent Company custom
synthesized and purified the phosphorothioated
oligonucleotide with the above sequence. In a previous
study, we have shown that this antisense
oligonucleotide blocks the effects of adenosine
on vascular smooth muscle cell
growth.8
Sense (5'-TTGGTCACTGGAACACGAG-3') and scrambled
(5'-GCAC- GCTCTATACTGCATG-3')
oligonucleotides were used as
controls.
Growth Studies
CFs were plated at a density of
5x103 cells/well in 24-well tissue culture
dishes and allowed to grow to subconfluence. Cells were then growth
arrested by feeding DMEM containing 0.25% albumin for 48 hours
in the presence or absence of 0.5 µmol/L of antisense, sense, or
scrambled oligonucleotides.
For 3H-thymidine incorporation (index of DNA synthesis), growth was initiated by treating growth-arrested cells for 20 hours with DMEM supplemented with fresh oligonucleotides and containing PDGF-BB (25 ng/mL) in the presence or absence of various treatments. After 20 hours of incubation, the treatments were repeated with freshly prepared solutions but supplemented with 3H-thymidine (1 µCi/mL). Four hours later, the experiments were terminated and 3H-thymidine in the trichloroacetic acid (10%)-precipitated fraction was counted in a liquid scintillation counter.8 For cell number experiments CFs were allowed to attach overnight, growth arrested for 48 hours and then treated every 24 hours for 4 days, and on day 5 cells were dislodged and counted with a Coulter counter.
3H-Proline incorporation studies were done to investigate the effects of various test agents on collagen synthesis. Confluent monolayers of CFs were made quiescent by feeding DMEM containing 0.25% albumin for 48 hours in the presence or absence of 0.5 µmol/L of antisense, sense, or scrambled oligonucleotides. Collagen synthesis were initiated by treating growth-arrested cells for 48 hours with DMEM supplemented with fresh oligonucleotides, PDGF-BB (25 ng/mL), and 3H-L-proline (1 µCi/mL) in the presence or absence of various treatments. Experiments were terminated by washing the cells twice with PBS and twice with ice-cold TCA (10%). The precipitate was solubilized and counted in a liquid scintillation counter.8 To make sure that the inhibitory effects of the experimental agents on collagen synthesis were not due to changes in cell number, the experiments were conducted in confluent monolayers of cells in which changes in cell number were precluded. Additionally, cell counting was performed in cells treated in parallel to the cells used for the collagen synthesis studies, and the data were normalized to cell number.
MAP Kinase Activity
CFs grown to confluence in
35-mm2 culture dishes were made quiescent by
feeding DMEM supplemented with 0.25% BSA and containing or lacking 0.5
µmol/L of antisense, sense, or scrambled
oligonucleotides for 48 hours. Growth-arrested CFs were
washed with PBS and pretreated for 24 hours with or without various
test agents in the presence or absence of fresh
oligonucleotides. Cells were then stimulated for 10
minutes with PDGF-BB (25 ng/mL). After stimulation, cells were washed
with ice-cold PBS and extraction buffer (50 mmol/L
ß-glycerophosphate, 1.5 mmol/L EGTA, 1 mmol/L
dithiothreitol, 100 µmol/L
Na3VO4, 10 µg/mL
aprotinin, 5 µg/mL pepstatin, 20 µg/mL leupeptin, and 1 mmol/L
benzamidine), scraped off the plates and sonicated for 20 seconds in
0.5 mL of extraction buffer. The extracts were collected, the cytosolic
fraction were separated by centrifuging the extracts at 100
000g for 20 minutes at 4°C,
and the supernatants were diluted to a concentration of 1 mg protein/mL
and were stored at -70°C for MAP kinase activity assays. The MAP
kinase activity in the cytosolic extracts was quantified by our
previously described method.9
Briefly, cytosolic extracts (5 µL) were added to 30 µL of MAP
kinase assay buffer [25 mmol/L ß-glycerophosphate, 1.25
mmol/L EGTA, 0.5 mmol/L dithiothreitol, 150 µmol/L
Na3VO4, 2 µmol/L
peptide inhibitor for cAMP-dependent protein kinase
(H-TTTAAPIAS-GATGAAAAI-NH2;
Bachem Bioscience Inc), 1 mg/mL bovine serum albumin, 10
µmol/L calmidizolium, 0.33 mg/mL myelin basic protein (MBP), and 100
µmol/L of [
32P]ATP]. After
incubation for 15 minutes at 30°C, 25-µL aliquots of the reaction
mixture were spotted onto phosphocellulose paper (Whatman), washed 4
times with 150 mmol/L phosphoric acid, and counted in 10 mL of
scintillation fluid on a gamma counter. To calculate the MAP kinase
activity, samples incubated in the absence of MBP were subtracted from
the same samples incubated with MBP.
cAMP Synthesis
Confluent monolayers of CFs were treated every 24
hours for 48 hours with or without 0.5 µmol/L of antisense, sense, or
scrambled oligonucleotide to A2B
receptor. After 48 hours, the monolayers were washed with PBS and CFs
treated for 30 minutes under standard tissue culture conditions with
PBS (buffered with HEPES, 25 mmol/L;
NaHCO3, 13 mmol/L) containing fresh
oligonucleotides and supplemented with 1 mmol/L
3-isobutyl-1-methylxanthine (IBMX) and containing or lacking 1 µmol/L
Cl-Ad. After treatment, the supernatants were collected and the cells
were treated with 1 mL of ice-cold propanediol. The extracellular (in
supernatant) and the intracellular (cellular fraction) cAMP levels were
then analyzed by high-performance liquid
chromatography with a previously described method. In
CFs grown in parallel and treated similarly, the monolayers were
inspected microscopically for intactness, the protein content was
measured, and the data were normalized to protein
content.
Statistics
All growth experiments were performed in triplicates
or quadruplicates with 3 to 4 separate cultures. Data are
presented as mean±SEM. Statistical analysis was
performed using ANOVA, paired Students’
t test, or Fisher’s Least
Significant Difference test as appropriate. A value of
P<0.05 was considered
statistically significant.
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Results |
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PDGF-BB significantly stimulated all measures of cell growth (DNA, collagen, and cell proliferation) by 7- to 10-fold. As shown in Figure 1, in PDGF-BB-treated cells, Cl-Ad inhibited in a concentration-dependent manner all measures of cell growth. The lowest concentration of Cl-Ad that significantly inhibited PDGF-BB-induced cell growth was 1 nmol/L, and at a concentration of 1 µmol/L, Cl-Ad inhibited cell growth by approximately 50%. High (10-6 mol/L), but not low, concentrations of CPA and CGS21680 inhibited PDGF-BB-induced cell growth (Figure 1). 5'-N-Ethylcarboxamidoadenosine (NECA) was more potent than AB-MECA, CPA, and CGS21680, but less potent than MECA or Cl-Ad in inhibiting PDGF-BB–induced increases cell growth (Figure 1). The inhibitory effects of Cl-Ad on all measures of cell growth were significantly blocked by KF17837 and DPSPX, but not by DPCPX (Figure 2). Similar to Cl-Ad, the inhibitory effects of MECA were blocked by KF17837 and DPSPX, but not by DPCPX (data not shown).
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EHNA plus IDO inhibited PDGF-BB–induced DNA synthesis, collagen synthesis, and cell proliferation (Figure 3). The inhibitory effects of EHNA plus IDO on PDGF-BB–induced cell growth were significantly attenuated by KF17837 and DPSPX, but not by DPCPX (Figure 3). Trypan blue exclusion tests indicated no loss in viability of cells treated with CPA, CGS21680, MECA, AB-MECA, KF17837, DPSPX, or DPCPX.
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Antisense, but not sense or scrambled, oligonucleotides increased basal as well as PDGF-BB–induced DNA synthesis by 52% and 56%, respectively. Consistent with our previous findings in vascular SMCs,8 preliminary studies showed that the stimulatory effect of the antisense oligonucleotide was biphasic in nature, and maximal stimulatory effects were observed at 0.2 to 0.5 µmol/L. Trypan blue exclusion and MTT assay showed no cell toxicity at 0.5 µmol/L. The inhibitory effects of Cl-Ad, MECA, EHNA plus IDO and cAMP (a precursor of adenosine) on PDGF-BB–induced cell proliferation, DNA synthesis, and collagen synthesis were completely abolished in CFs pretreated with antisense oligonucleotides, but not in CFs treated with sense or scrambled oligonucleotides (Figure 4).
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Treatment of CFs with PDGF-BB increased MAP kinase activity from 0.476±0.07 pmol/min per mg protein to 6.82±0.4 pmol/min per mg protein, and the stimulatory effects of PDGF-BB were inhibited by the MAP kinase inhibitor PD98059 (10 µmol/L) to 1.5±0.04 pmol/min per mg protein. The stimulatory effects of PDGF-BB on MAP kinase activity were inhibited in a concentration-dependent manner in CFs pretreated for 24 hours with 0.001 to 1 µmol/L of Cl-Ad. PDGF-BB-induced MAP kinase activity was also inhibited by 1 µmol/L of MECA, NECA, adenosine or EHNA plus IDO, but not in cells treated with CGS21680, AB-MECA, or CPA (Figure 5A). The inhibitory effects of Cl-Ad, and EHNA plus IDO were abrogated by KF17837 and DPSPX, but not by DPCPX (Figure 5A). Moreover, the inhibitory effects of adenosine were enhanced in the presence of EHNA plus IDO (Figure 5A). The inhibitory effects of Cl-Ad, MECA, cAMP, and EHNA plus IDO on PDGF-BB–induced MAP kinase activity were completely abolished in CFs pretreated with antisense, but not sense or scrambled, oligonucleotides (Figure 5B).
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Treatment of CFs with Cl-Ad induced intracellular and extracellular cAMP levels by 23.2-fold and 55.4-fold, respectively (Figure 6). The stimulatory effects of Cl-Ad on cAMP synthesis activity were completely abolished in CFs pretreated with antisense, but not sense or scrambled, oligonucleotides (Figure 6).
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Discussion |
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The present study demonstrates that A2B receptors mediate the antimitogenic effects of adenosine in cardiac fibroblasts. Treatment of CFs with a stable adenosine analog (Cl-Ad) and with agents that elevate endogenous adenosine (EHNA plus IDO) inhibited PDGF-BB–induced CF growth (DNA synthesis, collagen synthesis, cell proliferation, and MAP kinase activity). The potency of MECA, an adenosine agonist with high affinity for A2 receptors, was similar to Cl-Ad. The adenosine agonists CPA and CGS21680, which are selective A1 and A2A receptor agonists, respectively, were only weakly inhibitory at high concentration. Moreover, AB-MECA, an A3 adenosine receptor agonist, was ineffective in inhibiting PDGF-BB-induced CF growth. The inhibitory effects of Cl-Ad, MECA, EHNA plus IDO were significantly reversed by KF17837, a selective A2 receptor antagonist4 and by DPSPX, a nonselective A2 receptor antagonist,4 but not by DPCPX, a selective A1 receptor antagonist. Moreover, the inhibitory effects of MECA, Cl-Ad, and EHNA plus IDO on growth were completely abolished by antisense oligonucleotides to A2B receptors (to inhibit A2B receptor synthesis), but not by sense or scrambled oligonucleotides. These findings provide evidence that exogenous as well as CF-derived adenosine inhibits PDGF-BB–induced growth and MAP kinase activity in an autocrine/paracrine manner and by means of the A2B receptor.
That CPA (an adenosine analog that is highly selective for A1 receptors and mediates its effects at pharmacologically low doses [10-9 mol/L and lower]) does not inhibit PDGF-BB–induced growth at low concentrations suggests that the inhibitory effects of adenosine are not mediated by means of A1 receptors. This conclusion is further supported by our observation that DPCPX, an adenosine receptor antagonist that is a 700-fold selective for A1 receptors, does not block the inhibitory effects of Cl-Ad on growth. That KF17837 and DPSPX, but not DPCPX, attenuate the inhibitory effects of Cl-Ad suggest that the inhibitory effects of adenosine are A2 receptor mediated. That CGS21680 is ineffective in mimicking the inhibitory effects of adenosine and NECA is more effective than CGS21680 but less effective than Cl-Ad and MECA strongly supports the conclusion that the effects of adenosine are not mediated by means of A2A receptors. That AB-MECA, an A3 adenosine receptor agonist did not attenuate PDGF-BB–induced CF growth rules out the antimitogenic role for A3 adenosine receptor. Rather, our data suggests that A2B receptors mediate the inhibitory effects of adenosine on cell growth.
The hypothesis that the inhibitory effects of adenosine on growth are mediated by means of A2B receptors is supported further by the observation that the inhibitory effects of MECA and Cl-Ad on growth are completely abolished by antisense oligonucleotides, which inhibit the synthesis of A2B receptors. Importantly, neither sense nor scrambled oligonucleotides affect the inhibitory effects of MECA and Cl-Ad, which rules out nonspecific actions of the antisense oligonucleotides.
The above findings provide the first evidence that exogenous adenosine inhibits PDGF-BB–induced growth of CFs and that the inhibitory effects of adenosine are mediated by means of activation of A2B receptors. However, whether endogenous adenosine also inhibits CF growth cannot be inferred from studies with agonists. Therefore, we examined the growth inhibiting effects of agents that elevate cellular adenosine levels by means of different mechanisms to assess the role of endogenous, ie, CF-derived, adenosine on CF growth.
The physiological effects of adenosine are governed in part by the rapid rate of elimination of adenosine from the extracellular space. Metabolism of adenosine to inosine and AMP by adenosine deaminase and adenosine kinase, respectively,3 10 play a key role in the catabolism of active adenosine. Inhibition of the enzyme adenosine deaminase by EHNA and the enzyme adenosine kinase by IDO increases endogenous levels of adenosine.6 Treatment of CFs with EHNA plus IDO inhibits PDGF-BB–induced CF growth. That the inhibitory effects of EHNA plus IDO on CF growth are significantly reversed by KF17837 and DPSPX, but not DPCPX, provides evidence that the inhibitory effects of EHNA plus IDO are mediated by means of generation of adenosine and that these effects are A2 adenosine receptor mediated. Moreover, the observation that the growth inhibitory effects EHNA plus IDO are completely abolished in CFs pretreated with antisense oligonucleotides to A2B receptors, but not by sense or scrambled oligonucleotides, provides strong evidence that inhibitory effects of endogenous CF-derived adenosine are A2B receptor mediated.
One common signaling pathway that is activated by multiple growth factors and that is implicated in the cardiac remodeling process is the MAP kinase pathway.11 The MAP kinase pathway is activated at sites of ischemia-induced cardiac remodeling.11 MECA and Cl-Ad, but not CPA, AB-MECA and CGS21680, inhibit PDGF-BB–induced MAP kinase activity, and these effects are blocked by KF17837 and DPSPX, but not DPCPX, suggesting that inhibition of the MAP kinase pathway by means of A2B adenosine receptors contributes to the inhibitory effects of adenosine on cell growth. This idea is further supported by the observation that the inhibitory effects of MECA and Cl-Ad on PDGF-BB–induced MAP kinase activity are completely abolished by antisense oligonucleotides to A2B receptors, but not by sense or scrambled oligonucleotides. Moreover, treatment of CFs with inducers of endogenous adenosine (EHNA plus IDO) inhibits MAP kinase activity, and these effects are blocked by KF17837 and DPSPX, but not by DPCPX, suggesting that CF-derived adenosine inhibits MAP kinase activity by means of A2B receptors. This hypothesis is further supported by the observation that the inhibitory effects of EHNA plus IDO are completely abolished by antisense to A2B receptors, but not by sense or scrambled oligonucleotides. Importantly, the basal growth and MAP kinase activity of the CFs was significantly induced in CFs treated with antisense, suggesting that endogenously produced adenosine has inhibitory effects on MAP kinase activity and CF growth.
Although the above findings provide evidence that adenosine inhibits overall MAP kinase activity, these results may not accurately represent the activity of the p42 and p44 isoforms of MAP kinase. Because these MAP kinase isoforms were not immunoprecipitated from the lysates,4 participation of other kinases remains a possibility. In this regard, it is interesting to note that, in contrast to SMCs and CFs in endothelial cells, mast cells, and embryonic kidney-293 cells, A2B receptors increase, rather than inhibit, MAP kinase activity.4 Additional studies are needed to elucidate the different signal transduction pathways that our regulated by A2B receptors in various cell types.
That the inhibitory effects of exogenous cAMP on PDGF-BB-induced growth are abolished by antisense, but not sense or scrambled oligonucleotides, suggests that A2B receptors also play an important role in mediating the antimitogenic effects of exogenous cAMP. This idea is supported further by the findings that cAMP is a precursor of adenosine and CFs metabolize cAMP by means of an extracellular cAMP-adenosine pathway involving ecto-5'-nucleotidases,12 cAMP-derived adenosine inhibits CF growth by means of A2B receptors,12 and cAMP is hydrophilic and exogenous cAMP should not penetrate cell membranes. Therefore, the inhibitory effects of exogenous cAMP are largely mediated by means of adenosine and A 2B adenosine receptors.
That the growth inhibitory effects of exogenous and endogenous adenosine are abolished in CFs treated with antisense oligonucleotides, but not in CFs treated with sense or scrambled oligonucleotides, provides evidence that the A2B receptor expression is downregulated in CFs treated with antisense oligonucleotides. Because of unavailability of specific antibodies to A2B receptors, we were unable to confirm the downregulation by Western blotting or immunostaining. However, we did find that the effects of Cl-Ad on cAMP production, a signal transduction mechanism activated by A2B receptors, was abolished in CFs treated with antisense, but not in CFs treated with sense or scrambled oligonucleotides.
In contrast to antisense blockade of A2 receptors, neither DPSPX nor KF17837 increased basal or PDGF-BB–induced growth of CFs. There are several possible explanations for these results. DPSPX and FK17837 are not pure A2B receptor antagonists, but rather both also block A2A receptors, and DPSPX antagonizes A1 receptors as well. Antagonism of multiple adenosine receptors by DPSPX and KF17837 may explain the differential effects of antisense oligonucleotides versus receptor antagonists on basal cell growth. Alternatively, both DPSPX and KF17837 may have other nonspecific effects that affect cell growth. For example, DPSPX inhibits phosphodiesterase activity, and high concentrations (10-6 mol/L) of DPSPX directly inhibit cell growth. In contrast to receptor antagonists, antisense oligonucleotides downregulate A2B receptors; hence, other mechanisms influencing cell growth are not affected. Nonetheless, further studies using pure A2B receptor antagonists and agonists and molecular approaches are required to further explore the role A2B receptor in regulating cell growth.
In conclusion, we provide evidence that A2B, and not A1, A2A, or A3 receptors are responsible for mediating the antimitogenic effects of both exogenous and endogenous adenosine on PDGF-induced growth of vascular CFs. Thus, A2B receptors may play a vital role in regulating CF growth in health and disease. Moreover, abnormal/decreased expression of A2B receptors may contribute to abnormal synthesis and deposition of collagen and hypertrophy of CFs observed in CFs associated with hypertension, myocardial infarction, and reperfusion injury after ischemia.
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Acknowledgments |
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Supported by the Swiss National Science Foundation grant 32–54172.98 and National Institutes of Health grants HL-55314 and HL-35909.
Received October 25, 2000; first decision December 11, 2000; accepted December 19, 2000.
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References |
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 Y. Chen, S. Epperson, L. Makhsudova, B. Ito, J. Suarez, W. Dillmann, and F. Villarreal Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2478 - H2486. [Abstract] [Full Text] [PDF]
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I. Feoktistov, S. Ryzhov, H. Zhong, A. E. Goldstein, A. Matafonov, D. Zeng, and I. Biaggioni Hypoxia Modulates Adenosine Receptors in Human Endothelial and Smooth Muscle Cells Toward an A2B Angiogenic Phenotype Hypertension, November 1, 2004; 44(5): 649 - 654. [Abstract] [Full Text] [PDF]
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A. Afzal, L.C. Shaw, S. Caballero, P.E. Spoerri, A.S. Lewin, D. Zeng, L. Belardinelli, and M.B. Grant Reduction in Preretinal Neovascularization by Ribozymes That Cleave the A2B Adenosine Receptor mRNA Circ. Res., September 19, 2003; 93(6): 500 - 506. [Abstract] [Full Text] [PDF]
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 R. S. Ostrom, J. E. Naugle, M. Hase, C. Gregorian, J. S. Swaney, P. A. Insel, L. L. Brunton, and J. G. Meszaros Angiotensin II Enhances Adenylyl Cyclase Signaling via Ca2+/Calmodulin: Gq-Gs CROSS-TALK REGULATES COLLAGEN PRODUCTION IN CARDIAC FIBROBLASTS J. Biol. Chem., June 27, 2003; 278(27): 24461 - 24468. [Abstract] [Full Text] [PDF]
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 E. K. Jackson, C. Zhu, and S. P. Tofovic Expression of adenosine receptors in the preglomerular microcirculation Am J Physiol Renal Physiol, July 1, 2002; 283(1): F41 - F51. [Abstract] [Full Text] [PDF]
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E. K. Jackson and R. K. Dubey Role of the extracellular cAMP-adenosine pathway in renal physiology Am J Physiol Renal Physiol, October 1, 2001; 281(4): F597 - F612. [Abstract] [Full Text] [PDF]
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