A luciferase-engineered cell line for study of cAMP regulation in endothelial cells.
cAREL is a cAMP-responsive endothelial cell line carrying a luciferase reporter gene introduced by stable transfection of a luciferase enhancer trap into rabbit aortic endothelial cells. Luciferase gene expression in cAREL was stimulated 233-fold by 8-BrcAMP. Treatment with the beta-adrenoceptor agonist isoproterenol induced a 7.0-fold increase in luciferase expression, which was partially blocked by either beta1- or beta2-adrenoceptor antagonists and totally blocked by propranolol and by a combination of beta1- plus beta2-adrenoceptor antagonists. Receptor stimulation was mimicked by cholera toxin, forskolin, 8-BrcAMP, and isobutylmethylxanthine but not by 8BrcGMP, dexamethasone, or phorbol 12-myristate 13-acetate. Stimulation by isoproterenol was completely blocked by H-89, a protein kinase A inhibitor. cAREL was also stimulated by A-23187, and this effect was abrogated by EGTA and H-89. cAREL is the first cAMP-sensitive endothelial cell line described, and it can be useful as a positive control, as a model for cAMP regulation, as a background to genetic introduction of receptors, as an indicator of intracellular pathway activation, and as a tool to investigate cAMP effects on other signaling pathways.
Laboratório de Biologia Molecular, Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. xavier@helix.mgh.harvard.edu
J
J
Xavier-Neto
A C
AC
Pereira
A H
AH
Motoyama
J E
JE
Krieger
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013449:Sulfonamides; D014162:Transfection / Q000379:methods*
D000319:Adrenergic beta-Antagonists; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000001:Calcimycin; D002772:Cholera Toxin; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine