Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells.
OBJECTIVE
Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions.


METHODS
The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNgamma) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-kappaB) were studied by Western blot.


RESULTS
CARB increased NO synthesis by 57 +/- 5%, while a 150 +/- 10% increase in NO liberation was triggered by LPS plus IFNgamma treatment. CARB added to LPS plus IFNgamma potentiated NO synthesis by 227 +/- 19%. CARB also upregulated NOS1 protein expression via NF-kappaB activation. In addition CARB and LPS plus IFNgamma stimulated PGE(2) synthesis by 72 +/- 9 and 42 +/- 4%, respectively, while CARB added to LPS plus IFNgamma treated cells produced a synergism in PGE(2) liberation (130 +/- 12%) via COX-2.


CONCLUSIONS
Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.
Español
A J
AJ
Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 piso 16, Buenos Aires, CP 1121, Argentina.
Goren
N
N
Ribeiro
M L
ML
Sales
María Elena
ME
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects; D041701:Swiss 3T3 Cells
D008070:Lipopolysaccharides; D018721:Muscarinic Agonists; D016328:NF-kappa B; D011976:Receptors, Muscarinic; D009569:Nitric Oxide; D007371:Interferon-gamma; D002217:Carbachol; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D051546:Cyclooxygenase 2; D015232:Dinoprostone