adenosine-5--(n-ethylcarboxamide) and butaprost

Pulmonary fibroblasts regulate extracellular matrix production and degradation and are critical in maintenance of lung structure, function, and repair, but they also play a central role in lung fibrosis. cAMP-elevating agents inhibit cytokine- and growth factor-stimulated myofibroblast differentiation and collagen synthesis in pulmonary fibroblasts. In the present study, we overexpressed adenylyl cyclase 6 (AC6) in pulmonary fibroblasts and measured cAMP production and collagen synthesis. AC6 overexpression enhanced cAMP production and the inhibition of collagen synthesis mediated by isoproterenol and beraprost, but not the responses to butaprost or PGE(2). To examine if increased AC6 expression would impact the development of fibrosis in an animal model, we generated transgenic mice that overexpress AC6 under a fibroblast-specific promoter, FTS1. Lung fibrosis was induced in FTS1-AC6(+/-) mice and littermate controls by intratracheal instillation of saline or bleomycin. Wild-type mice treated with bleomycin showed extensive peribronchial and interstitial fibrosis and collagen deposition. By contrast, FTS1-AC6(+/-) mice displayed decreased fibrotic development, lymphocyte infiltration (as determined by pathological scoring), and lung collagen content. Thus, AC6 overexpression inhibits fibrogenesis in the lung by reducing pulmonary fibroblast-mediated collagen synthesis and myofibroblast differentiation. Because AC6 overexpression does not lead to enhanced basal or PGE(2)-stimulated levels of cAMP, we conclude that endogenous catecholamines or prostacyclin is produced during bleomycin-induced lung fibrosis and that these signals have antifibrotic potential.

Topics: Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Alprostadil; Animals; Bleomycin; Calcium-Binding Proteins; Catecholamines; Colforsin; Collagen; Cyclic AMP; Epoprostenol; Fibroblasts; Isoproterenol; Lung; Membrane Microdomains; Mice; Mice, Transgenic; Pulmonary Fibrosis; Rats; S100 Calcium-Binding Protein A4; S100 Proteins; Signal Transduction

1. The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2. 2. PGE2 (0.001-10 microM) inhibited FMLP (100 nM)-induced O2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC50 of 0.15 +/- 0.03 microM, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3. The EP2-receptor agonists, misoprostol, 11-deoxy PGE1, AH13205 and butaprost, all at 10 microM, inhibited O2- generation, causing 95.5 +/- 2.9%, 56.8 +/- 5.2%, 37.1 +/- 6.6% and 18.9 +/- 4.4% inhibition respectively, the latter two being much less effective than PGE2. Similarly, the EP1-receptor agonist, 17-phenyl PGE2 (10 microM), and the EP3/EP1-receptor agonist, sulprostone (10 microM), also inhibited O2- generation, causing 32.2 +/- 7.0% and 15.3 +/- 3.4% inhibition respectively. 4. The non-selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 +/- 5.0%. In addition, IBMX shifted concentration-effect curves for PGE2, misoprostol, 11-deoxy PGE1, butaprost, and AH 13205 to the left, to give EC50s of 0.04 +/- 0.03 (n = 13), 0.07 +/- 0.03 (n = 4), 0.08 +/- 0.03 (n = 4), 0.33 +/- 0.13 (n = 4) and 0.41 +/- 0.2 microM (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated. This agrees well with the relative potencies of these agonists at EP2 receptors.5. By contrast, even in the presence of IBMX (0.25 mM), sulprostone and 17-phenyl PGE2 were only effective at the highest concentration (10 microM), and gave EECs of > 700 and 486 respectively, suggesting that EP1 or EP3 receptors are not involved.6. The selective type IV phosphodiesterase inhibitor, rolipram at 2 and 10 nM did not inhibit the FMLP response, but at the higher concentration of 50 nM, it decreased the FMLP response by 46.6 +/-7.3%.However, rolipram shifted concentration-effect curves for PGE2 to the left to give EC50s of 0.06 +/-0.022,0.015 +/- 0.0, 0.012 +/- 0.006 microM at 2, 10 and 50 nM respectively, compared to the control EC50 of0.27+/- 0.09 microM for PGE2.7. The EP4/TP receptor blocking drug, AH 23848B (10 microM, 1

Topics: Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Alprostadil; Cyclic AMP; Dinoprostone; Dose-Response Relationship, Drug; Humans; Misoprostol; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Prostaglandin E; Superoxides