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(Circulation Research. 2004;94:353.)
© 2004 American Heart Association, Inc.

Cellular Biology

Interleukin-1ß, Transforming Growth Factor-ß₁, and Bradykinin Attenuate Cyclic AMP Production by Human Pulmonary Artery Smooth Muscle Cells in Response to Prostacyclin Analogues and Prostaglandin E₂ by Cyclooxygenase-2 Induction and Downregulation of Adenylyl Cyclase Isoforms 1, 2, and 4

H. El-Haroun, D. Bradbury, A. Clayton, Alan J. Knox

From the Division of Respiratory Medicine, University of Nottingham, City Hospital, Nottingham, UK.

Correspondence to Prof Alan J. Knox, Division of Respiratory Medicine, University of Nottingham, Clinical Science Building, City Hospital, Nottingham, NG5 1PB, England. E-mail alan.knox{at}nottingham.ac.uk

	Abstract

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Increased levels of inflammatory cytokines contribute to the pathophysiology of pulmonary hypertension. Prostacyclin (PGI₂) analogues, which relax pulmonary vessels mainly through cAMP elevation, have a major therapeutic role. In this study, we show that prolonged incubation with bradykinin (BK), interleukin-1ß (IL-1ß), and transforming growth factor-ß₁ (TGF-ß₁) markedly impairs cAMP accumulation in human pulmonary artery smooth muscle cells in response to short-term incubation with prostaglandin E₂ (PGE₂) and the PGI₂ analogues iloprost and carbaprostacyclin. A similar reduction in cAMP accumulation in response to a direct adenylyl cyclase activator, forskolin, suggested that the effect was attributable to downregulation of adenylyl cyclase. Reverse transcriptase–polymerase chain reaction studies showed downregulation of adenylyl cyclase isoforms 1, 2, and 4. The effect of IL-1ß, BK, and TGF-ß₁ on cAMP levels was abrogated by the selective COX-2 inhibitor NS398. Furthermore, it was mimicked by prolonged incubation with the COX-2 product PGE₂ and PGI₂ analogues or the COX substrate arachidonic acid, suggesting that it was mediated by endogenous prostanoids produced by COX-2. Consistent with this, IL-1ß, BK, and TGF-ß₁ all induced COX-2 and PGE₂ release. These results show that BK, IL-1ß, and TGF-ß₁ downregulate adenylyl cyclase in human pulmonary artery smooth muscle cells via COX-2 induction and prostanoid release. This suggests a novel mechanism whereby mediators and cytokines produced in pulmonary hypertension may impair the therapeutic effects of prostacyclin analogues such as iloprost and carbaprostacyclin.

Key Words: interleukin-1ß • transforming growth factor-ß₁ • bradykinin • cAMP • adenylyl cyclase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prostacyclin (PGI₂) analogues are effective treatment for pulmonary hypertension.¹ Long-term treatment with intravenous PGI₂ improved survival rates and reduced vascular resistance in primary and secondary pulmonary hypertension.² PGI₂ analogues act mainly through cAMP, a relaxant second messenger,³ by binding to adenylyl cyclase–coupled prostacyclin receptors.^4–6 PGI₂ analogues also regulate pulmonary artery remodeling.⁷ Administration of PGI₂ analogues may compensate for defective PGI₂ production in pulmonary hypertension. Pulmonary vascular tone and remodeling is controlled by the balance between vasoconstrictor and vasodilator mediators.⁸ In pulmonary hypertension, there is an imbalance with excess thromboxane A₂ and reduced dilator PGI₂ production⁹ and pulmonary artery PGI₂ synthase expression.¹⁰ Endothelin-1 (ET-1) plays an important role in pulmonary hypertension. ET-1 levels are elevated in patients with pulmonary hypertension. ET-1 has a growth regulatory effect on pulmonary smooth muscle cells, partly via K⁺ channels.¹¹

Inflammatory cytokines and mediators contribute to the increased pulmonary resistance and remodeling in pulmonary hypertension,¹² including interleukin-1 (IL-1), IL-6, ET-1, and prostanoids.^13,14 IL-1ß is interesting because its elevated levels correlate with disease severity¹³ and prolonged IL-1ß stimulation regulates the adenylyl cyclase cascade in several cell systems. IL-1ß reduced cAMP production in response to isoproterenol in human airway smooth muscle cells,¹⁵ adult rat cardiac fibroblasts,¹⁶ and rat lung membranes.¹⁷ Although most studies have focused on ß₂ adrenoceptor signaling via adenylyl cyclase, they are relevant to prostacyclin signaling. The effect of IL-1ß on ß₂-adrenoceptor signaling is usually upstream of adenylyl cyclase at the receptor¹⁷ or G protein coupling.¹⁸

Other cytokines and mediators relevant to pulmonary hypertension also regulate adenylyl cyclase. Transforming growth factor (TGF)-ß has three isoforms, TGF-ß₁, TGF-ß₂, and TGF-ß₃.¹⁹ In primary pulmonary hypertension (PPH), TGF-ß immunoreactivity is increased in muscular arteries.²⁰ Pulmonary artery smooth muscle cells (PASMCs) from patients with PPH proliferate abnormally in response to TGF-ß₁.²¹ It is notable that prolonged TGF-ß₁ incubation reduces adenylyl cyclase activity and ß₂-adrenoceptor numbers in human embryonic lung fibroblasts.²²

Interest is also being shown in the role of bradykinin (BK) in pulmonary hypertension, because a BK antagonist prevented pulmonary hypertension in hypoxic rats.²³ BK also interferes with ß₂-adrenoceptor agonist–induced cAMP accumulation.²⁴ Thus, data show that IL-1ß, bradykinin, and TGF-ß₁ can interfere with ß₂-adrenoceptor–linked cAMP accumulation. Whether these agents interfere with prostacyclin-mediated cAMP accumulation in any biological system is unknown.

In this study we tested the hypothesis that IL-1ß, TGF-ß₁, or BK would impair cAMP generation in response to prostacyclin analogues in PASMCs. Such an effect would reduce the effect of PGI₂ analogues in pulmonary hypertension. We found that IL-1ß, TGF-ß₁, and BK reduced cAMP accumulation in response to prostacyclin analogues or prostaglandin E₂ (PGE₂). Mechanistic studies suggested that this effect was attributable to induction of COX-2, the inducible form of cyclooxygenase, with subsequent generation of prostanoids, because it was mimicked by exogenous PGI₂, PGE₂, or arachidonic acid and inhibited by the selective COX-2 inhibitor NS398. Forskolin studies suggested a direct effect on adenylyl cyclase, and reverse transcriptase–polymerase chain reaction (RT-PCR) showed downregulation of adenylyl cyclase isoforms 1, 2, and 4. These studies are the first in any biological system to show that IL-1ß, BK, and TGF-ß₁ impair cAMP generation in response to PGI₂ analogues. This has important implications for the use of PGI₂ and its analogues in pulmonary hypertension.

	Materials and Methods

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Cell Culture
Human proximal PASMCs (passage 3) from a 22-year-old man were purchased from Clonetics (BioWittaker UK Ltd, Wokingham, Berkshire, UK) and cultured to passage 6 in smooth muscle cell growth medium-2 Bulletkit (Clonetics BioWittaker UK Ltd).

Experimental Protocols
Cells were cultured to confluence in smooth muscle cell growth medium in humidified 5% CO₂ and 95% air at 37°C in 24-well plates and growth arrested in serum-free medium for 24 hours before experiments. Media were replaced with fresh serum-free medium containing cytokines or mediators, IL-1ß (10 ng/mL per 24 hours), BK (10 µmol/L per 24 hours), and TGF-ß₁ (1 ng/mL per 36 hours) and incubated for indicated periods. Experiments using PGE₂ and arachidonic acid (AA) were conducted similarly. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulphonamide (NS398) was added 30 minutes before cytokines when studied. NS398 was dissolved in dimethyl sulfoxide, and all other agents were dissolved in serum-free medium.

cAMP Assay
Media were removed and cells washed three times with PBS. cAMP accumulation was measured as previously described.¹⁵ Cells were incubated in 0.5-mL fresh medium plus 1 mmol/L 3 isobutyl-1-methylxanthene (IBMX), a phosphodiesterase inhibitor, and stimulated with carbaprostacyclin, iloprost, PGE₂, or forskolin (FSK) for 20 minutes. cAMP generation was terminated with 0.1 mL 30% (wt/vol) ice-cold trichloroacetic acid followed by amine/Freon extraction.²⁵ cAMP content was determined by protein binding assay.²⁶ Bound [³H] cAMP was measured using the Tri-Carb 2100TR liquid scintillation analyser (Packard Bioscience Ltd). cAMP levels were calculated using RiaSmart software (Packard Bioscience Ltd).

PGE₂ Assay
PGE₂ levels were measured by radioimmunoassay as described.^27,28

Western Blot Analysis
Western blotting for COX-1 and COX-2 was performed as described.²⁹ The goat anti mouse horseradish peroxidase was purchased from BD Biosciences (Cowley).

RNA Isolation and Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was isolated using the RNeasy mini kit (Qiagen). Total RNA 1 µg was reverse transcribed in a total volume of 25 µL including 200 U of M-MLV RT (Promega), 25 U of RNase inhibitor (Promega), and 0.5 µg of oligo (dT)₁₅ primer, 0.5 mmol/L of each dNTP, 1x first-strand buffer (Promega). The reaction was incubated at 42°C for 90 minutes.³⁰ PCR was performed by adding 12 µL of cDNA to 40 µL reaction mixture, giving final concentrations of 1 mmol/L MgCl₂, 0.12 mmol/L of each dNTP, 1 U of Taq polymerase, 1x PCR buffer (Promega), and 0.5 µmol/L of both the upstream and downstream PCR primers (Sigma) to all 9 adenylyl cyclase isoforms, as described by Xu et al³¹ (online Table 1, available at http://www.circresaha.org). Amplification was carried out with a PTC-100 programmable thermal controller (MJ Research Inc) after an initial denaturation at 95°C for 2 minutes. GAPDH was used as an internal control gene. This was followed by 40 cycles of PCR: denaturation at 95°C for 30 seconds, primer annealing at 52°C for 1 minute (isoforms 1, 2, and 8), 50°C for 1 minute (isoforms 3), 55°C for 1 minute (isoforms 4, 5, and 7), and 58°C for 1 minute (isoforms 6 and 9), primer extension at 68°C for 1 minute, and a final extension of 68°C for 10 minutes.³¹ PCR products were electrophoresed on 2% agarose gel in 0.5x TBE buffer containing 0.5 µg/mL ethidium bromide and visualized using ultraviolet illumination and GeneGenius gel documentation and analysis system (Syngene).³⁰

Materials
Recombinant human IL-1ß and recombinant human TGF-ß₁ were purchased from R&D Systems Europe Ltd, and bradykinin (BK), PGE₂, FSK, isoproterenol (Iso), AA, IBMX, trichloroacetic acid, and amine/Freon extraction were from Sigma. NS398 and carbaprostacyclin were purchased from Cayman Chemical (Alexis Corporation). Iloprost was a gift from Schering (Burgess Hill, West Sussex, UK). Protein kinase A–dependent cAMP and cAMP were from Sigma, and [8-³H] cAMP (specific activity 962GBq/mmol/L) was from Amersham Life Science (Little Chalfont).

Statistical Analysis
cAMP levels were measured in quadruplicate wells, and experiments were repeated at least three times. Data were expressed as mean and SEM and analyzed with Graph Pad Prism version 4.0 (Graph Pad software). Comparisons were by one-way ANOVA with the Tukey post hoc test. P<0.05 was regarded as statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PGI₂ Analogues, PGE₂, and Forskolin but not Isoproterenol Increase cAMP Production
Basal level of cAMP was 0.37±0.59 pmol/100 µL. Ten-micromolar PGI₂ analogues (carbaprostacyclin and iloprost), 10 µmol/L PGE₂, or 10 µmol/L FSK increased cAMP accumulation, reaching a peak within 20 to 30 minutes (online Figure 1). There was no significant change in cAMP levels in cells treated with isoproterenol (data not shown).

IL-1ß, BK, and TGF-ß₁ Reduce Carbaprostacyclin-Induced cAMP Production
Basal cAMP level was low in both untreated cells and cells pretreated with BK, IL-1ß, and TGF-ß₁. Carbaprostacyclin (0.01, 0.1, 1, 10 µmol/L) concentration dependently increased cAMP. IL-1ß (10 ng/mL) or BK (10 µmol/L) pretreatment for 24 hours (Figures 1A and 1B) significantly attenuated cAMP formation in response to all carbaprostacyclin (carbaprost) concentrations (P<0.001). TGF-ß₁ (1 to 10 ng/mL) pretreatment for 24 hours had no significant effect on cAMP production (data not shown). However, treatment with TGF-ß₁ (1 ng/mL) for 36 hours significantly attenuated cAMP production with all carbaprostacyclin concentrations (P<0.001) compared with cells without TGF-ß₁ pretreatment (Figure 1C).

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of IL-1ß, BK, and TGF-ß1 on cAMP production in response to carbaprostacyclin (carbaprost). Confluent human PASMCs were incubated with IL-1ß (10 ng/mL per 24 hours) (A), BK (10 µmol/L per 24 hours) (B), or TGF-ß1 (1 ng/mL per 36 hours) (C), the medium was removed, and cells were incubated with 0.1 mmol/L IBMX for 10 minutes. Carbaprost was added at the concentrations indicated for 20 minutes. cAMP was measured by protein-binding assay. Each bar is the mean of 8 determinations from 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001 compared with cells without IL-1ß, BK, and TGF-ß1 pretreatment.

IL-1ß, BK, and TGF-ß₁ Reduce Iloprost-Induced cAMP Production
We next determined if we would see similar effects using another prostacyclin analogue. Iloprost (0.01, 0.1, 1, and 10 µmol/L) markedly increased cAMP production concentration dependently. Pretreatment with IL-1ß (10 ng/mL) and BK (10 µmol/L) for 24 hours or TGF-ß₁ (1 ng/mL) for 36 hours significantly attenuated iloprost-induced cAMP production (online Figures 2A, 2B, and 2C).

IL-1ß, BK, and TGF-ß₁ Reduce PGE₂-Induced cAMP Generation
PGE₂ also generates cAMP by binding to EP₂ and EP₄ receptors. PGE₂-like PGI₂ is a major COX product in PASMCs. We therefore determined whether IL-1ß, BK, and TGF-ß₁ would downregulate PGE₂-mediated cAMP generation. Stimulation with PGE₂ concentration dependently increased cAMP generation. IL-1ß (10 ng/mL per 24 hours) pretreatment attenuated cAMP formation in response to 0.1, 1, and 10 µmol/L PGE₂ (P<0.05, P<0.001, and P<0.01, respectively; Figure 2A). BK (10 µmol/L per 24 hours) and TGF-ß₁ (1 ng/mL per 36 hours) significantly attenuated cAMP formation in response to 0.1, 1, and 10 µmol/L PGE₂ (P<0.001; Figures 2B and 2C).

View larger version (24K): [in this window] [in a new window]   Figure 2. Concentration response of PGE2 on cAMP generation from untreated PASMCs and cells pretreated with IL-1ß, BK, and TGF-ß1. After preincubation with IL-1ß (10 ng/mL per 24 hours) (A), BK (10 µmol/L per 24 hours) (B), or TGF-ß1 (1 ng/mL per 36 hours) (C), the medium was removed and cells were incubated with 0.1 mmol/L IBMX for 10 minutes. PGE2 was added at the concentrations indicated for 20 minutes. Values are expressed as mean±SEM of 8 determinations of 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001.

IL-1ß, BK, and TGF-ß₁ Reduce FSK-Induced cAMP Production
To determine whether the effect was on adenylyl cyclase, we used the direct adenylyl cyclase activator FSK rather than prostanoids to generate cAMP. FSK concentration-dependently increased cAMP. IL-1ß (10 ng/mL) or BK (10 µmol/L) treatment for 24 hours reduced cAMP generation in response to 1 and 10 µmol/L FSK (Figures 3A and 3B, respectively). TGF-ß₁ (1 ng/mL) for 36 hours significantly reduced cAMP production only in response to the highest concentration of FSK (10 µmol/L) but not other concentrations compared with cells without TGF-ß₁ pretreatment (Figure 3C).

View larger version (19K): [in this window] [in a new window]   Figure 3. Concentration response of FSK on cAMP generation from PASMCs pretreated with IL-1ß, BK, and TGF-ß1 and cells without pretreatment. After preincubation with IL-1ß (10 ng/mL per 24 hours) (A), BK (10 µmol/L per 24 hours) (B), or TGF-ß1 (1 ng/mL per 36 hours) (C), the medium was removed and cells were incubated with 0.1 mmol/L IBMX for 10 minutes. FSK was added at the concentrations indicated for 20 minutes. Values are expressed as mean±SEM of 8 determinations of 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001.

Effect of IL-1ß, BK, and TGF-ß₁ on Specific Adenylyl Cyclase Isoform mRNA
To determine whether IL-1ß, BK, and TGF-ß₁ were downregulating specific adenylyl cyclase isoforms, we used RT-PCR to detect mRNA to all 9 adenylyl cyclase isoforms. Isoforms 1, 2, 3, 4, 6, 7, and 9 were expressed in human PASMCs and IL-1ß or BK incubation for 24 hours, and TGF-ß₁ for 36 hours significantly reduced adenylyl cyclase isoform 1, 2, and 4 mRNA (Figures 4A and 4B) but had no effect on other isoforms (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 4. Effect of IL-1ß, BK, and TGF-ß1 on adenylyl cyclase isoform mRNA. Confluent PASMCs were incubated with IL-1ß (10 ng/mL per 24 hours), BK (10 µmol/L per 24 hours), or TGF-ß1 (1 ng/mL per 36 hours). Adenylyl cyclase isoform and GAPDH (as internal control) mRNA were measured by RT-PCR (A). Relative densities were calculated by dividing the density of adenylyl cyclase isoform bands by the GAPDH bands at the same point (B).

Role of COX-2 in IL-1ß–Induced, BK-Induced, and TGF-ß₁–Induced Downregulation of cAMP Formation
We have previously shown that IL-1ß, BK, and TGF-ß₁ all induce COX-2 in PASMCs.³² To determine whether endogenous prostanoids produced as a result of COX-2 induction were responsible for the downregulation of adenylyl cyclase, we studied the effect of the selective COX-2 inhibitor NS398 (1 µmol/L). The reductions in carbaprostacyclin, iloprost, PGE₂, and FSK-induced cAMP accumulation in response to IL-1ß, BK, and TGF-ß₁ were all significantly attenuated by NS398 (Figures 5A through 5D).

View larger version (38K): [in this window] [in a new window]   Figure 5. Effect of NS398 on IL-1ß–induced, BK-induced, and TGF-ß1–induced changes in cAMP in response to carbaprostacyclin (carbaprost) (A), iloprost (B), PGE2 (C), or FSK (D). Cells were pretreated with or without 1 µmol/L NS398 for 30 minutes before incubation with IL-1ß (10 ng/mL per 24 hours), BK (10 µmol/L per 24 hours), or TGF-ß1 (1 ng/mL per 36 hours). Cells were treated with 1 µmol/L carbaprostacyclin, iloprost, PGE2, or FSK plus 1 mmol/L IBMX for 20 minutes. Each bar is mean of 8 determinations from 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001 compared with cells without pretreatment.

IL-1ß, BK, and TGF-ß₁ Induce COX-2 Protein Expression and Increase PGE₂ Production With Inhibition of PGE₂ by Pretreatment With NS398
To confirm that IL-1ß, BK, and TGF-ß₁ were inducing COX-2, we performed Western blots. Western blotting showed maximum induction of COX-2 protein in response to 10 ng/mL IL-1ß, 10 µmol/L BK at 2 hours, and 1 ng/mL TGF-ß₁ at 8 hours (Figure 6A). Incubation with IL-1ß or BK for 24 hours and TGF-ß₁ for 36 hours increased PGE₂ release (Figure 6B), which was significantly inhibited by NS398 (1 µmol/L) pretreatment (Figure 6C).

View larger version (30K): [in this window] [in a new window]   Figure 6. Time course of IL-1ß, BK, and TGF-ß1 effects on COX-2 induction in PASMCs (A). B, Effect of IL-1ß, BK, and TGF-ß1 on PGE2 production by PASMCs for 24, 24, and 36 hours, respectively. C, Effect of NS398 on IL-1ß–induced, BK-induced, and TGF-ß1–induced PGE2 production. Each bar is the mean of 8 determinations from 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001.

Effect of Exogenous PGI₂ Analogues on cAMP Generation in Response to Carbaprostacyclin, Iloprost, PGE₂, and FSK
If endogenous prostanoids are responsible for the effect of IL-1ß, BK, and TGF-ß₁, then exogenous prostanoids should mimic their effect. Consistent with this, exogenous PGI₂ analogue (iloprost, 1 µmol/L) pretreatment for 24 hours markedly attenuated cAMP generation in response to carbaprostacyclin, iloprost, PGE₂, or FSK in a concentration-dependent fashion (carbaprostacyclin and iloprost, P<0.01 for all concentrations; 0.1 and 1 µmol/L PGE₂, P<0.01; 10 µmol/L, P<0.001; and 1 and 10 µmol/L FSK, P<0.05 and <0.001, respectively) compared with the cells without PGI₂ pretreatment (Figures 7A through 7D). This provides additional evidence that COX-2 products are involved in attenuating cAMP accumulation to receptor-linked cAMP stimulants after IL-1ß, BK, and TGF-ß₁ treatment.

View larger version (39K): [in this window] [in a new window]   Figure 7. Effect of exogenous PGI2 analogue (iloprost) on cAMP generation in response to carbaprostacyclin (A), iloprost (B), PGE2 (C), or FSK (D). After preincubation without or with 1 µmol/L PGI2 analogue for 24 hours, cells were treated with increasing concentrations of carbaprostacyclin, iloprost, PGE2, or FSK plus 1 mmol/L IBMX for 20 minutes. Each bar is the mean of 8 determinations from 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001 compared with cells without PGI2 pretreatment.

Effect of Exogenous PGE₂ on cAMP Generation in Response to Carbaprostacyclin, Iloprost, PGE₂, and FSK
PGE₂ pretreatment (1 µmol/L) for 24 hours markedly attenuated cAMP generation in response to carbaprostacyclin, iloprost, PGE₂, and FSK concentration dependently (carbaprost and iloprost, P<0.01; 0.1 and 1 µmol/L PGE₂, P<0.01; 10 µmol/L, P<0.001; and 1 and 10 µmol/L FSK, P<0.05 and <0.001, respectively) compared with cells without PGE₂ pretreatment (online Figures 3A through 3D). These results provide additional evidence that COX-2 products are responsible for the impaired cAMP responses caused by BK, IL-1ß, and TGF-ß₁.

Effect of COX Substrate AA on cAMP Formation in Response to Carbaprostacyclin, Iloprost, PGE₂, and FSK
To additionally explore the role of COX products, we determined if the COX substrate AA would reduce cAMP generation induced by carbaprostacyclin, iloprost, PGE₂, and FSK. AA (1 µmol/L) for 24 hours significantly attenuated cAMP generation in response to all concentrations of carbaprostacyclin and iloprost (P<0.001; Figures 8A and 8B), 0.1 and 1 µmol/L PGE₂ (P<0.01), 10 µmol/L PGE₂ (P<0.05) (Figure 8C), and 1 and 10 µmol/L FSK (P<0.05 and P<0.001, respectively; Figure 8D) compared with cells without AA pretreatment.

View larger version (41K): [in this window] [in a new window]   Figure 8. Effect of exogenous AA on cAMP generation in response to carbaprostacyclin (A), iloprost (B), PGE2 (C), or FSK (D). After preincubation without or with 1 µmol/L AA for 24 hours, cells were treated with increasing concentrations of carbaprostacyclin, iloprost, PGE2, or FSK plus 1 mmol/L IBMX for 20 minutes. Each bar is the mean of 8 determinations from 3 independent experiments, where *P<0.05, **P<0.01, and ***P<0.001 compared with cells without AA pretreatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings in these studies are that prolonged pretreatment of human PASMCs with IL-1ß, BK, and TGF-ß₁ attenuated carbaprostacyclin-, iloprost-, and PGE₂-induced cAMP accumulation. Furthermore, the attenuated response was prevented by the COX-2–selective inhibitor NS398 and mimicked by exogenous PGE₂ and PGI₂ analogues or the COX substrate AA, providing strong evidence that the effect was mediated by induction of COX-2 and prostanoid generation. We have previously shown that, as here, IL-1ß, BK, and TGF-ß₁ all induce COX-2 in PASMCs.³² Studies here with FSK suggested that IL-1ß, BK, and TGF-ß₁ were acting on adenylyl cyclase. Consistent with this, RT-PCR studies showed downregulation of adenylyl cyclase isoforms 1, 2, and 4. These findings have important implications for the treatment of pulmonary hypertension because they imply that cytokines and mediators may downregulate the response to PGI₂ and its analogues.

During development of pulmonary hypertension, the pulmonary circulation becomes less responsive to vasodilators, and structural remodeling of pulmonary arteries occurs.¹¹ cAMP is important in the control of pulmonary vascular tone³³ and remodeling.^34,35 cAMP-mediated pulmonary vascular smooth muscle relaxation can be accomplished by different receptor-linked pathways. Prostanoids (mainly PGE₂ and PGI₂) activate prostaglandin EP₂ and EP₄ and prostacyclin receptors, which are coupled via G stimulatory proteins (G_s) to adenylyl cyclase to generate cAMP.^4,36–38 cAMP then activates cAMP-dependent protein kinase A,³⁷ which phosphorylates several intracellular substrates to cause relaxation. PGI₂ also inhibits pulmonary artery smooth muscle proliferation in part through cAMP accumulation and activation of protein kinase A.^7,39

Both PGI₂ analogues we tested and PGE₂ stimulated cAMP synthesis. The small differences in their responses most likely reflect their slightly different pharmacology. We studied two analogues to reinforce our findings. Their effects were broadly similar. PGI₂ and PGE₂ are major COX products in human PASMCs.^32,40 In contrast, isoproterenol did not increase cAMP, suggesting that these cells have few ß₂-adrenoceptors. Few previous studies have looked at the effects of isoproterenol on pulmonary artery smooth muscle, although in rats isoproterenol induced pulmonary artery relaxation through cAMP.⁴ The contrast between our study and the others may reflect species differences or perhaps loss of ß₂-adrenoceptors in culture.

Cyclooxygenase converts arachidonic acid into prostaglandin H₂, which is converted to prostanoids by specific synthases. Three isoforms of COX exist.⁴¹ COX-1 produces physiological prostanoid levels,⁴² whereas COX-2 is induced in inflammatory conditions.⁴³ COX-3 is a splice variant of COX-1 whose function is unclear.⁴⁴ COX products, particularly those of COX-2, regulate several cellular processes involved in inflammation and remodeling, including angiogenesis, chemokine production, apoptosis, and matrix metalloproteinase production.^{15,24,29,45–47}

Because COX products can regulate adenylyl cyclase function,⁴⁵ we hypothesized that this phenomenon might occur in PASMCs and regulate cAMP responses to agents acting on adenylyl cyclase–coupled receptors. To test this hypothesis, we used three agents that induce COX-2 in human PASM cells, namely IL-1ß, BK, and TGF-ß₁. We found that all three agents reduced cAMP accumulation to PGI₂ analogues and PGE₂, although the effect with TGF-ß₁ was only apparent after more prolonged incubation. This is consistent with the potency and time course of COX-2 induction in this and in our previous studies, where TGF-ß₁ was the weakest inducer of COX-2, which acted more slowly.³² An increase in COX-2 protein expression is seen after 2 and also at 8 hours with both IL-1ß and BK but is not apparent until 8 hours with TGF-ß₁. To test the role of COX-2 products in the cAMP response attenuation, we studied the effect of NS398, a selective COX-2 inhibitor. The concentration of NS398 we used selectively inhibits COX-2–mediated prostanoid generation by >90%.⁴⁸ Consistent with a major role for COX-2, NS398 abolished the effect of all three agents on prostanoid-mediated cAMP accumulation. The role of COX products was additionally established by showing that exogenously applied PGI₂ analogues, PGE₂, or the COX substrate arachidonic acid mimicked the effect of IL-1ß, BK, and TGF-ß₁.

It is recognized that G protein–coupled receptors can become desensitized or downregulated during chronic stimulation. This has been extensively studied with ß₂-adrenoceptors⁴⁹ but can also be seen with PGI₂ receptors.³⁸ This can be agonist-specific homologous desensitization, which usually occurs by receptor downregulation or effects on G protein coupling⁵⁰ or nonagonist-specific heterologous desensitization mediated by changes in adenylyl cyclase or downstream phosphodiesterases.⁵¹

There are thus several sites in the adenylyl cyclase signaling system whereby chronic cytokine and inflammatory mediator stimulation by releasing prostanoids could attenuate cAMP levels in response to subsequent stimulation with PGI₂ analogues. Because we used IBMX,¹⁵ we can exclude a role for phosphodiesterases in our experiments. Our studies with forskolin suggest that the major site of attenuation of cAMP responses was adenylyl cyclase. To test this hypothesis additionally, we used RT-PCR to measure mRNA of the 9 known isoforms of adenylyl using primers previously described.³¹ Under resting conditions, human PASMCs expressed isoforms 1, 2, 3, 4, 6, 7, and 9, giving bands of the expected molecular weights. IL-1ß, BK, and TGF-ß₁ consistently downregulated adenylyl cyclase isoforms 1, 2, and 4. Isoforms 3, 6, 7, and 9 were unchanged. This suggests that downregulation of isoforms 1, 2, and 4 is the mechanism responsible. These studies are the first to identify the adenylyl cyclase isoforms present in human PASMCs and differ from the rat, where isoforms 2, 3, 5, 6, 7, and 8 were found.⁵²

The findings with IL-1ß, BK, and TGF-ß₁ contrast with results in other biological systems, although most studies have focused on ß₂-adrenoceptor rather than prostanoid signaling. In tracheal smooth muscle, IL-1ß attenuated relaxation to isoproterenol via induction of the inhibitory G protein subunits G_i2 and G_i3.⁵³ In rat cardiac fibroblasts, IL-1ß attenuated cAMP accumulation via PDE2.¹⁶ IL-1ß upregulated PDE4 in human myometrial cells.⁵⁴ Conversely, IL-1ß upregulated ß₂-adrenoceptors in human airway epithelial cells,⁵⁵ suggesting complex cell-specific effects. Our studies are the first in any biological system to show that IL-1ß, BK, and TGF-ß₁ impair cAMP generation in response to PGI₂ analogues. Our cells were derived from pulmonary conduit vessels, and it would be interesting to determine if the same is true in smaller-resistance vessels.

In conclusion, we show that IL-1ß, BK, and TGF-ß₁ downregulate adenylyl cyclase and attenuate cAMP generation in response to PGI₂ analogues and PGE₂ in human PASMCs. We provide strong evidence that COX-2 induction and prostanoid release play a critical role in this process and that it involves downregulation of adenylyl cyclase isoforms 1, 2, and 4. This would be expected to result in impaired PASMC relaxation to PGI₂ and its analogues in pulmonary hypertension.

	Acknowledgments

 
We thank the Egyptian High Commission for supporting Dr El-Haroun and the Medical Research Council (UK) for supporting Dr Clayton.

	Footnotes

 
Original received April 25, 2003; resubmission received October 10, 2003; revised resubmission received November 18, 2003; accepted November 26, 2003.

	References

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