ABSTRACT
AIMS: The present study aimed to explore possible effects of endothelin-1 (ET-1) on ß(2)-adrenoceptor gene transcription in human lung fibroblasts. MAIN METHODS: MRC-5 human lung fibroblasts were cultured in absence or presence of test substances, followed by ß(2)-adrenoceptor mRNA determination by quantitative real time PCR. KEY FINDINGS: ET-1 caused a marked and rapid in onset (1 hr) increase in ß(2)-adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the ß(2)-adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on ß(2)-adrenoceptor mRNA was prevented by the non-selective ET-A/ET-B receptor antagonist bosentan, indicating that it was mediated via specific ET receptors. In the presence of actinomycin D the effect of ET-1 was prevented indicating that ET-1 acts via increased transcription of the ß(2)-adrenoceptor gene. ET-1-induced up-regulation of ß(2)-adrenoceptor mRNA was also seen in the presence of cycloheximide excluding indirect effects via up-regulation of other regulatory proteins. CONCLUSIONS: ET-1 can up-regulate ß-adrenoceptor gene transcription in human lung fibroblasts.
Subject(s)
Endothelin-1/physiology , Fibroblasts/metabolism , Transcription, Genetic , Up-Regulation , Benzoxazines/pharmacology , Bosentan , Cell Line , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Endothelin A Receptor Antagonists , Endothelin B Receptor Antagonists , Fibroblasts/drug effects , Humans , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptor, Endothelin A/metabolism , Receptor, Endothelin B/metabolism , Receptors, Adrenergic, beta-2/drug effects , Receptors, Adrenergic, beta-2/genetics , Sulfonamides/pharmacology , Time FactorsABSTRACT
Airway remodeling is a structural alteration associated with chronic inflammatory and obstructive airway diseases, wherein fibroblasts are crucially involved. The present study investigates whether lung fibroblast proliferation is influenced by muscarinic mechanisms. For this purpose, expression of muscarinic receptors in MRC-5 human lung fibroblasts was characterized by semiquantitative RT-PCR, and the effects of muscarinic agonists and antagonists on ((3)H)-thymidine incorporation as a measure of proliferative activity were studied under different culture conditions. MRC-5 fibroblasts express mRNA encoding different subtypes of muscarinic receptors (M(2) > M(3) > M(4), traces for M(5) and no M(1)). Expression of M(2) and M(3) receptors was confirmed at the protein level by immunoblot analysis. Under different culture conditions, carbachol (up to 10 microM) or oxotremorine (10 microM) stimulated ((3)H)-thymidine incorporation, with maximum increases between about 40 and 100%. The stimulatory effect of 10 microM carbachol was prevented by pretreatment with pertussis toxin and antagonized in a concentration-dependent manner by the muscarinic receptor antagonists tiotropium, AQ-RA 741, AF-DX 384, 4-diphenylacetoxy-N-methylpiperidine methoiodide, himbacine, p-fluorohexahydrosiladifenidol, and pirenzepine, with concentrations producing 50% inhibition of 14 pM, 24, 64, 127, 187, 452 nM, and 1.5 microM, respectively. Primary human lung fibroblasts were also found to express mRNA for muscarinic receptors (M(2) > M(1) > M(3), traces for M(4) and no M(5)), and showed a pertussis toxin-sensitive proliferative response to muscarinic receptor stimulation. In conclusion, proliferation of human lung fibroblasts can be stimulated by activation of muscarinic receptors with a pharmacologic profile correlating best to M(2) receptors.
Subject(s)
Cell Proliferation , Fibroblasts/metabolism , Lung/cytology , Receptors, Muscarinic/metabolism , Cell Line , Dose-Response Relationship, Drug , Humans , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , RNA, Messenger/metabolism , Receptor, Muscarinic M2/drug effects , Receptor, Muscarinic M2/metabolism , Receptors, Muscarinic/geneticsABSTRACT
Effects of phosphodiesterase inhibitors on L-arginine-dependent pathways in rat alveolar macrophages, inducible nitric oxide (NO) synthase (iNOS) and arginase, were studied. Culture of rat alveolar macrophages in the presence of lipopolysaccharides (20 h) caused an increase of arginase activity (by 135%) and nitrite concentration (fourfold). The nonselective phosphodiesterase inhibitor IBMX (2-isobutyl-1-methylxanthine) enhanced arginase activity by 35% and nitrite accumulation by 130%. IBMX caused a clear increase in iNOS protein levels and a relatively smaller increase in iNOS mRNA. The effect of IBMX on nitrite accumulation was largely attenuated by the protein kinase A inhibitor K 5720. The phosphodiesterase 4 inhibitor rolipram enhanced nitrite accumulation more effectively than the phosphodiesterase 3 inhibitor siguadozan (about 50% and 20% of IBMX effect, respectively), whereas the phosphodiesterase 3/4 inhibitor benzafendrine was as effective as IBMX. In conclusion, in rat alveolar macrophages, phosphodiesterase 4 and, to a smaller extent, phosphodiesterase 3 play a role in the control of iNOS-mediated NO synthesis.
