(Hypertension. 2001;37:1095.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Endogenous Cyclic AMP-Adenosine Pathway Regulates Cardiac Fibroblast Growth
From the Center for Clinical Pharmacology, Departments of Medicine (R.K.D., D.G.G., Z.M., E.K.J.) and Pharmacology (E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, Pa; and the Clinic for Endocrinology (R.K.D.), Department of Obstetrics and Gynecology, University Hospital Zurich, Switzerland.
Correspondence to Dr Raghvendra K. Dubey, Center for Clinical Pharmacology, 623 Scaife Hall, 200 Lothrop St, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582. E-mail dubey{at}novell2.dept-med.pitt.edu
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Abstract |
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Abstract—Our previous studies show that cardiac fibroblasts express the extracellular "cAMP-adenosine pathway," that is, the generation of adenosine from extracelluar cAMP. The goal of this study was to assess whether activation of the cAMP-adenosine pathway by stimulation of endogenous cAMP synthesis regulates cardiac fibroblast growth. Cardiac fibroblasts in 3D cultures were used as the model system. Treatment of cardiac fibroblasts with forskolin, isoproterenol, or norepinephrine increased cAMP production and extracellular levels of adenosine, and these effects were prevented by inhibition of adenylyl cyclase (2',5'-dideoxyadenosine). Treatment with forskolin, isoproterenol, or norepinephrine for 24 hours inhibited DNA synthesis (3H-thymidine incorporation), and this effect was enhanced by combined inhibition of adenosine deaminase (erythro-9-[2-hydroxy-3-nonyl] adenine) plus adenosine kinase (iodotubercidin). Inhibition of adenylyl cyclase or adenosine receptors (1,3-dipropyl-8-p-sulfophenylxanthine or KF17837) prevented the effects of forskolin, isoproterenol, and norepinephrine on DNA synthesis. Forskolin also inhibited protein synthesis (3H-leucine incorporation) and cell proliferation, and these effects were blocked by adenosine receptor antagonism. Treatment of cardiac fibroblasts with norepinephrine for >48 hours but not <48 hours increased DNA synthesis, protein synthesis, and cell number. However, blockade of adenylyl cyclase or antagonism of adenosine receptors caused norepinephrine to induce proliferation in <48 hours. Our findings indicate that the endogenous cAMP-adenosine pathway regulates cardiac fibroblast growth.
Key Words: adenosine • cyclic AMP • fibroblasts • myocardial infarction • hypertension, experimental • remodeling
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Introduction |
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Recent studies in our laboratory demonstrate that extracellular cAMP is an important determinant of adenosine production in cardiac fibroblasts (CFs) through a biochemical mechanism we refer to as the cAMP-adenosine pathway.1 This pathway involves the conversion of extracellular cAMP to AMP and hence to adenosine by the enzymes ecto-phosphodiesterase (ecto-PDE) and ecto-5'-nucleotidase (ecto-5'-NT), respectively. Our previous studies also indicate that exogenous adenosine and exogenous cAMP, through metabolism to adenosine, potently inhibit CF growth.1 2 However, whether stimulation of the endogenous cAMP-adenosine pathway inhibits CF growth through adenosine formation is unknown. The purpose of this study was to assess whether CF-derived cAMP regulates CF growth through activation of the cAMP-adenosine pathway. To achieve our goal, we evaluated the effects of forskolin, isoproterenol, and norepinephrine, agents that stimulate adenylyl cyclase, on fetal calf serum (FCS)-induced DNA synthesis, cell proliferation, and protein synthesis in 3D cultures of ventricular CFs. Moreover, we evaluated whether the effects of these agents on cell growth were reduced by inhibition of adenylyl cyclase and antagonism of adenosine receptors and enhanced by inhibition of adenosine metabolism. Moreover, we assessed whether the growth effects of these agents were accompanied with changes in cAMP and adenosine levels.
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Methods |
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CF Cell Culture
Hearts were harvested from pentobarbital-anesthetized (50 mg/kg IP), male Sprague-Dawley rats; the left ventricular CFs were isolated and cultured as previously described by us.2 For all the studies, the method of Mio et al,3 with minor modifications, was then used to grow ventricular CFs in 3D cultures. Briefly, 0.8 mL of 2 mg/mL collagen (type I from rat tail; Sigma Chemical Co) solution in 0.5% acetic acid was mixed at 4°C with 0.21 mL of dilution medium (455 µL 10x basal medium, 112 µL of 7.5% NaHCO3, 50 µL of 200 mmol/L L-glutamine, 13 µL of FCS, 25 to 50 µL of 0.142 mol/L NaOH to adjust pH to 7.5, and 425 µL [minus vol of NaOH] of H2O). Subsequently, 0.3 mL of collagen mixture, devoid of air bubbles, was plated in each well of a 24-well culture plate and allowed to gel for 1 hour at 37°C. CFs were then plated on the top layer and allowed to adhere by incubation under standard tissue culture conditions. After overnight incubation, the medium was aspirated and a fresh layer (250 µL) of collagen mixture was layered on top of the CFs and allowed to gel for 20 minutes at 37°C. Cells were treated with 1 mL of DMEM/F-12 supplemented with 10% FCS; cells were allowed to grow to subconfluence for growth studies or to confluence for the cAMP and adenosine synthesis studies. Experiments were conducted in CFs in the second or third passage. All chemicals used for tissue culture were from GIBCO Laboratories. KF17837 was kindly provided by Dr Fumio Suzuki of Hakko Kogyo Co Ltd (Sunto, Shizuoka, Japan). Forskolin, isoproterenol, and norepinephrine were purchased from Sigma, and the source of all other chemicals was as previously described.2
Adenosine and cAMP Synthesis
Studies
CFs were washed twice with HEPES-buffered Hanks
balanced salt solution and treated with 0.5 mL of Dulbecco’s PBS
buffered with HEPES [25 mmol/L] and
NaHCO3 [13 mmol/L] in the presence and
absence of various treatments. After 30 minutes, the supernatant was
collected and frozen at -70°C until adenosine, inosine, and
AMP levels were measured. The remaining cells were dislodged by
treating cultures with 0.5 mL of a mixture of collagenase
(1 mg/mL; Sigma) and trypsin (0.25%), and the number of cells in each
well were counted with a Coulter counter. To ensure that the various
treatments caused no toxic effects or cell death, trypan blue exclusion
assays were used to evaluate the viability of CFs treated in parallel.
Adenosine, AMP, and inosine levels in the samples were
analyzed by high-pressure liquid
chromatography, with our previously described
method.4 The concentration of
each purine in the samples was calculated from a standard curve and
normalized to cell number.
Growth Studies
3H-Thymidine and
3H-leucine incorporation studies were
performed to investigate the effects of treatments on FCS-induced DNA
and total protein synthesis, respectively. CFs
(104 cells/well) were plated in 24-well
tissue culture dishes and allowed to grow in DMEM/F-12 containing 10%
FCS under standard tissue culture conditions. For
3H-thymidine incorporation, subconfluent CFs
were growth-arrested with culture medium containing 0.25% FCS for 48
hours and subsequently treated for 20 hours with culture medium
supplemented with 2.5% FCS in the presence and absence of various
treatments. After 20 hours, the treatments were repeated with freshly
prepared solutions but supplemented with
3H-thymidine (1 µCi/mL, NEN) for an
additional 4 hours. For 3H-leucine
incorporation, confluent CFs were growth-arrested for 48 hours and
treated for 48 hours with culture medium containing 2.5% FCS and
3H-leucine (1 µCi/mL, ICN Biochemicals) in
the presence and absence of various treatments. Cells were washed twice
with Dulbecco’s PBS, dislodged by digestion with 0.5 mL of a mixture
of collagenase (1 mg/mL) and trypsin (0.25%), and treated
with 10% ice-cold trichloroacetic acid. The trichloroacetic
acid–precipitated cell pellet was obtained by
centrifugation and was solubilized in 0.5 mL of 0.3N
NaOH and 0.1% sodium dodecyl sulfate. Aliquots from 4 wells
for each treatment, with 10 mL scintillation fluid, were counted in a
liquid scintillation counter. Each experiment was conducted in
triplicate or quadruplicate and repeated 3 to 5 times.
To evaluate the effects of treatments on cell proliferation, growth-arrested cells were incubated in the presence and absence of various treatments in DMEM supplemented with 2.5% FCS. Treatment was repeated after 48 hours, and cell counts were assayed after 4 days of treatment by dislodging cells with 0.5 mL of a mixture of collagenase (1 mg/mL) and trypsin (0.25%) and counting cells in a Coulter counter.
Statistics
All experiments were conducted in triplicate or
quadruplicate and repeated 3 to 4 times with separate cultures. Results
are presented as mean±SEM. Statistical analyses were
performed with ANOVA. When evaluating a treatment-dependent effect
and/or concentration-dependent effect within a group, data were
analyzed by 1-factor ANOVA followed by Fisher’s least
significant difference test for multiple comparisons. As appropriate, a
Bonferroni t test or Dunnett
multiple comparison test was applied to compare differences between
groups. All treatment-related effects within a group at a specific time
point were compared by Student’s unpaired
t test. A value of
P<0.05 was considered
statistically significant.
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Results |
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Forskolin and isoproterenol significantly inhibited FCS-induced 3H-thymidine incorporation in a concentration-dependent manner (Figure 1), and the inhibitory effects of forskolin and isoproterenol were significantly attenuated by 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, A1/A2 adenosine receptor antagonist) and KF17837 (A2 adenosine receptor antagonist) but not by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, A1 adenosine receptor antagonist). Forskolin significantly inhibited FCS-induced cell proliferation, and this effect also was attenuated significantly by DPSPX and KF17837 but not DPCPX (Figure 2). Treatment of CFs for 24 hours with norepinephrine inhibited FCS-induced 3H-thymidine incorporation, and this effect was attenuated by DPSPX (Figure 3 and 4). 2',5'-Dideoxyadenosine (DDA, adenylyl cyclase inhibitor) significantly attenuated the inhibitory effects of forskolin, isoproterenol, and norepinephrine on 3H-thymidine incorporation (Figure 3) and the effects of forskolin on cell proliferation (Figure 2). Forskolin inhibited 3H-leucine incorporation by 47±4%, and this effect was attenuated significantly by DPSPX and DDA (Figure 3). Both norepinephrine and isoproterenol significantly stimulated FCS-induced 3H-leucine incorporation in CFs, and this effect was significantly enhanced by DPSPX and DDA (Figure 3).
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P<0.01 compared with NE, FOR, or ISO alone.
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Norepinephrine and isoproterenol inhibited 3H-thymidine incorporation but stimulated 3H-leucine incorporation. A possible explanation for these contrasting effects of norepinephrine and isoproterenol is that 3H-thymidine incorporation was assessed after 24 hours of treatment, whereas 3H-leucine incorporation was determined after 48 hours of treatment. To investigate this possibility, the time course of norepinephrine effects on growth were studied by treating CFs with norepinephrine for 24 to 96 hours. Treatment with norepinephrine (100 nmol/L) induced time-dependent effects on CF growth (Figure 4). Treatment of CFs with norepinephrine for <48 hours and >48 hours inhibited and stimulated, respectively, FCS-induced 3H-thymidine incorporation and cell number (Figure 4). Both DDA and DPSPX reversed the early inhibitory effects of norepinephrine and significantly enhanced the late stimulatory effects of norepinephrine (Figure 4).
Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; 10 µmol/L; adenosine deaminase inhibitor) plus iodotubercidin (IDO; 0.1 µmol/L; adenosine kinase inhibitor) significantly inhibited FCS-induced 3H-thymidine incorporation and enhanced the effects of forskolin and isoproterenol on 3H-thymidine incorporation (Figure 5). Furthermore, DPSPX but not DPCPX significantly reversed the inhibitory effects of forskolin and isoproterenol in the presence and absence of EHNA plus IDO (Figure 5).
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As shown in Figure 6, cAMP levels increased significantly in the medium of CFs treated with forskolin, isoproterenol, and norepinephrine (379±26, 126±7, and 128±4 nmol/L/106 cells, respectively); however, adenosine levels in the medium were unchanged. Because extracellular adenosine is rapidly metabolized, we assayed the effects of forskolin, isoproterenol, and norepinephrine on cAMP and adenosine levels in the presence of EHNA plus IDO. Treatment of CFs with EHNA+IDO increased the levels of adenosine and cAMP in the medium from 0.6±0.18 to 241±10 nmol/L/106 cells and below detection limit to 0.543±0.13 nmol/L/106 cells. Also, in the presence of EHNA+IDO, isoproterenol, forskolin and norepinephrine increased both cAMP and adenosine levels in the medium. Forskolin-, isoproterenol-, and norepinephrine-induced cAMP synthesis was significantly inhibited in the presence of DDA. A low concentration of DPSPX (Figure 6) did not inhibit cAMP and adenosine synthesis induced by forskolin, isoproterenol, or norepinephrine (Figure 6). Also, forskolin-, isoproterenol-, and norepinephrine-induced cAMP and adenosine synthesis were not blocked by DPCPX or KF17837. The cAMP and adenosine levels in CFs treated in presence of EHNA+IDO with forskolin, forskolin+DPCPX, and forskolin+KF17837 were 498±20, 488±26, and 513±18 nmol/L/106 cells, respectively, for cAMP and 388±23, 394±18, and 367±26 nmol/L/106 cells, respectively, for adenosine. The cAMP and adenosine levels in cells treated with isoproterenol and norepinephrine were 164±14 and 275±24 nmol/L/106 cells, respectively, for isoproterenol and 191±12 and 355±20 nmol/L/106 cells, respectively, for norepinephrine. Similar to forskolin, the stimulatory effects of isoproterenol and norepinephrine on cAMP and adenosine synthesis were not blocked by DPCPX or KF17837 and influenced by <2% to 4% (P>0.05).
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Discussion |
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Several lines of evidence suggest that adenosine plays a major role as an endogenous nucleoside that protects the heart and the vasculature against cardiovascular disease. Formation of adenosine within the heart and/or near the blood vessel wall is believed to occur through three biochemical pathways.5 The intracellular ATP pathway and extracellular ATP pathway entail dephosphorylation of ATP to adenosine inside and outside the cell, respectively. The transmethylation pathway involves the hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine by the enzyme S-adenosyl-L-homocysteine–hydrolase. Because the intracellular and extracellular ATP pathways of adenosine production require crisis events and the transmethylation pathway is mostly constitutive, the three traditional routes of adenosine biosynthesis are not well suited for physiological modulation. More recently, we have identified a fourth pathway, the cAMP-adenosine pathway, for adenosine production.6 7 8 9 10 In contrast to the other pathways for adenosine formation, the cAMP-adenosine pathway may be more amenable to physiological modulation by hormones. Because a large amount of adenosine is synthesized in the heart, we hypothesize that the cAMP-adenosine pathway may also be present in cardiac cells and more specifically in CFs, which constitute 60% of the total heart cells.
The activation of adenylyl cyclase turns on the cAMP-adenosine pathway, which has both intracellular and extracellular sites of adenosine production. Intracellular or extracellular metabolism of cAMP to AMP and AMP to adenosine is catalyzed through cytosolic PDE and ecto-PDE, respectively, and cytosolic 5'-nucleotidase and ecto-5'-nucleotidase, respectively. However, intracellular formation of adenosine may be diminished by the competition of cytosolic 5'-nucleotidase and adenylate kinase for AMP and by the competition of transport mechanisms with adenosine kinase for adenosine. Therefore, the extracellular limb of the cAMP-adenosine pathway may be quantitatively more important.
In a recent study, we provided evidence for the physiological relevance of the cAMP-adenosine pathway in regulating CF growth.1 In this regard, our previous study shows that the inhibitory effects of cAMP on FCS-induced CF growth are significantly enhanced in the presence of EHNA, which prevents the metabolism of adenosine to inosine, plus IDO, which inhibits the metabolism of adenosine to AMP. Moreover, the inhibitory effects of cAMP but not Br-cAMP (stable cAMP analog) are significantly reversed in the presence of KF17837 and DPSPX. Because the inhibitory effects of exogenous as well as endogenous adenosine on smooth muscle cell and CF proliferation are mediated by A2 receptors,8 11 12 the fact that KF17837 and DPSPX but not DPCPX block the inhibitory effects of cAMP on CF growth, whereas KF17837, DPSPX, and DPCPX do not reverse the inhibitory effects of Br-cAMP, indicates that the cAMP-adenosine pathway may contribute importantly to the regulation of cardiac biology and more specifically CF proliferation.
Although our previous findings provide evidence that
adenosine derived from exogenous cAMP inhibits CF growth,
whether similar effects are exerted
physiologically by endogenously
generated cAMP is unclear. To address this issue, we evaluated the
effects of agents known to induce cellular cAMP levels on the growth of
CFs. The findings that forskolin increases cAMP and adenosine
levels in the medium, inhibits FCS-induced DNA synthesis, cell
proliferation, and protein synthesis, and that these effects are
blocked by the adenylyl cyclase inhibitor DDA as well as by
A2-adenosine receptor
antagonists KF17837 and DPSPX provide convincing evidence
that cAMP-derived adenosine inhibits CF growth and that these
effects are A2-adenosine receptor
mediated. This notion is further strengthened by the observation that
the effects of DDA are accompanied by decreases in the levels of cAMP
and adenosine, whereas DPSPX and KF17837 do not reduce the
exogenous levels of adenosine. Similar to forskolin, both
isoproterenol and norepinephrine induce cAMP and
adenosine synthesis and inhibit FCS-induced DNA synthesis in
CFs. Also like forskolin, the effects of isoproterenol and
norepinephrine on cAMP and adenosine synthesis and
DNA synthesis are abolished by the adenylyl cyclase
inhibitor DDA, and the effects of isoproterenol and
norepinephrine on DNA synthesis are attenuated by the
A2 adenosine receptor
antagonists DPSPX and KF17837. The observation that in
contrast to forskolin, isoproterenol and norepinephrine
induce protein synthesis, even though all three agents increase cAMP
and adenosine levels and inhibit DNA synthesis, does not
disprove the inhibitory role of cAMP on growth. Indeed,
several studies report similar effects for norepinephrine
and isoproterenol on the growth of cardiac cells. In addition to
inducing cAMP, catecholamines increase the synthesis of
several mitogenic factors such as endothelin,
angiotensin II, and transforming growth
factor-
.13 14
Because the net result on growth depends on the balanced generation of
growth inducers and growth inhibitors, it is feasible that
as compared with cAMP, norepinephrine and isoproterenol
induce the generation of hypertrophic factors much more. In this
regard, it is plausible that the observed increases in protein
synthesis in response to 48-hour treatment with
catecholamines are due to delayed mitogenic
effects of catecholamines. This hypothesis is supported by
our observations that (1) norepinephrine inhibits
FCS-induced DNA synthesis and cell number in CFs treated for <48 hours
but induces CF growth in CFs treated for >48 hours; and (2) the
stimulatory effects of norepinephrine and isoproterenol on
protein synthesis and the delayed mitogenic effects of
norepinephrine on DNA synthesis and cell number are
significantly enhanced when cAMP generation is inhibited by DDA or when
adenosine effects are blocked with DPSPX. Taken together, the
above findings confirm that the net effects of cAMP on CF growth are
inhibitory.
The above findings support the hypothesis that the cAMP-adenosine pathway is of physiological relevance in maintaining homeostasis within the cardiovascular system. The feasibility of this system is also evident from the fact that vascular endothelial and smooth muscle cells, as well as heart cells (myocytes, endothelial cells, and fibroblast cells), are well-equipped with ecto-5'-NT, a ubiquitous enzyme that efficiently metabolizes AMP to adenosine and constitutes a part of the extracellular limb of the cAMP-adenosine pathway.15 Because activation of adenylyl cyclase always causes egress of cAMP into the extracellular space,16 provided that sufficient levels of ecto-PDE exist, activation of adenylyl cyclase would trigger the extracellular metabolism of cAMP to AMP and hence to adenosine. Because these reactions would take place in a highly localized environment, this newly formed adenosine could then act in an autocrine and/or paracrine fashion to modulate (amplify, inhibit, and/or expand) the local response to hormonal stimulation of adenylyl cyclase. It is important to note that relatively modest increases in cAMP production could give rise to significant concentrations of adenosine at the cell surface because adenosine would be synthesized by a series of spatially linked enzymatic reactions.
Abnormal growth of CFs contributes to the structural changes in the heart associated with hypertension and myocardial infarction, and this adversely affects the performance of the heart. A balanced basal production of growth-promoting and growth-inhibiting factors maintains homeostasis within the heart and prevents pathological cardiac remodeling. Our previous studies show that CFs and smooth muscle cells synthesize adenosine and that exogenous as well as endogenous (cell-derived) adenosine inhibits FCS-induced growth in these cells in an autocrine/paracrine fashion.11 12 On the basis of the observation that CFs have the capability of efficiently converting cAMP to adenosine, it is feasible that if the rate of cAMP production is increased in response to hormonal stimulation, CFs can effectively increase the extracellular levels of adenosine. Moreover, the increased generation of adenosine locally at the surface of CFs may be an important mechanism by which CFs protect themselves against factors generated to induce abnormal growth of CFs and cardiac remodeling.
Summary
These experiments provide the first evidence that CFs
are capable of metabolizing endogenous cAMP to
adenosine through the cAMP-adenosine pathway and that
adenosine generated from endogenous cAMP inhibits
FCS-induced growth of ventricular CFs in an
autocrine/paracrine fashion. Moreover, our studies demonstrate that
norepinephrine stimulates the cAMP-adenosine
pathway and that this contributes to the delayed mitogenic
effects of norepinephrine on ventricular CFs.
Therefore, adenosine produced by the metabolism of
cAMP by CFs may play a vital role in cardiac physiology/cell biology,
and abnormalities in the cAMP-adenosine pathway may contribute
importantly to the abnormal proliferation of CFs observed in
hypertension and myocardial
infarction.
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Acknowledgments |
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This study was supported by the Swiss National Science Foundation grant 32–54172.98 and National Institutes of Health grants HL-55314 and HL-35909.
Received May 25, 2000; first decision June 21, 2000; accepted September 29, 2000.
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