Abstract-Increased levels of inflammatory cytokines contribute to the pathophysiology of pulmonary hypertension.Prostacyclin (PGI 2 ) 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-1␤ (IL-1␤), and transforming growth factor-␤ 1 (TGF-␤ 1 ) markedly impairs cAMP accumulation in human pulmonary artery smooth muscle cells in response to short-term incubation with prostaglandin E 2 (PGE 2 ) and the PGI 2 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-1␤, BK, and TGF-␤ 1 on cAMP levels was abrogated by the selective COX-2 inhibitor NS398. Furthermore, it was mimicked by prolonged incubation with the COX-2 product PGE 2 and PGI 2 analogues or the COX substrate arachidonic acid, suggesting that it was mediated by endogenous prostanoids produced by COX-2. Consistent with this, IL-1␤, BK, and TGF-␤ 1 all induced COX-2 and PGE 2 release. These results show that BK, IL-1␤, and TGF-␤ 1 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-1␤ Ⅲ transforming growth factor-␤ 1 Ⅲ bradykinin Ⅲ cAMP Ⅲ adenylyl cyclase P rostacyclin (PGI 2 ) analogues are effective treatment for pulmonary hypertension. 1 Long-term treatment with intravenous PGI 2 improved survival rates and reduced vascular resistance in primary and secondary pulmonary hypertension. 2 PGI 2 analogues act mainly through cAMP, a relaxant second messenger, 3 by binding to adenylyl cyclase-coupled prostacyclin receptors. 4 -6 PGI 2 analogues also regulate pulmonary artery remodeling. 7 Administration of PGI 2 analogues may compensate for defective PGI 2 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 A 2 and reduced dilator PGI 2 production 9 and pulmonary artery PGI 2 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 ϩ 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-1␤ is interesting because its elevate...

Here we tested the effect of interleukin-1␤, a proinflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the ␤ 2 adrenoreceptor and EP prostanoid receptors. Interleukin-1␤ significantly increased isoprenaline-induced cAMP accumulation by increasing ␤ 2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1␤ significantly impaired prostaglandin E 2 -induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E 2 production, and a resulting downregulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1␤ increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E 2 . Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP 4 receptors, but not EP 2 , and receptor expression is reduced by interleukin-1␤. Collectively, these results provide mechanistic insight into how interleukin-1␤ can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.

We have previously shown that interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1beta, BK, and TGF-beta1 reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Galphai protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Galphas protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1beta, BK, and TGF-beta1. These studies suggest that IL-1beta, BK, and TGF-beta1 impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.