Treatment with LPS plus INF‐γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

Abstract

BACKGROUND AND PURPOSELPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. EXPERIMENTAL APPROACHNIH3T3 cells were treated with LPS (10 ng·mL ) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. KEY RESULTSThe cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. CONCLUSIONS AND IMPLICATIONSInflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.

Escherichia coli -induced inflammation changes the expression of acetylcholine receptors (M2R, M3R, and α-7 nAChR) in the pig uterus

“…In the myometrium of bacteria-treated uteri, upregulation of M3R protein expression and downregulation of α-7 nAChR protein expression were revealed. The current data are partly consistent with reports showing reduced expression of M2R and increased expression of M3R in inflammatory states of the airways in the course of chronic obstructive pulmonary disease (27), induction of expression of M3R and M5R in murine fibroblasts in response to inflammation (6), and greater expression of α-7 nAChR in severe preeclampsia placentas (18). In the present experiment, only the changes in the inflamed endometrial M3R mRNA expression in relation to the Ctrl and Sal groups were coincident with the product of this gene.…”

“…The factors that cause the changes in M2R, M3R, and α-7 nAChR expression might include lipopolysaccharide (LPS) released from bacteria ( 22 ) and cytokines (pro-inflammatory) produced in large amounts ( 8 , 34 ) in uterine infection. It is known that tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) synergise to reduce the M2R expression in fibroblasts of human embryonic lung (HEL 299 cells) ( 10 ) and that LPS and interferon-γ induce expression of M3R and M5R in murine fibroblasts ( 6 ). Moreover, the mAChR level in the current experiment may have been modulated by the steroid hormones, which were present in the rat myometrium under physiological conditions ( 36 ).…”

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

“…Proliferation was evaluated by using the soluble tetrazolium salt 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay (Life Technologies, Argentina) as previously described . In living cells, MTT is reduced to formazan.…”

“…Proliferation was evaluated by using the soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Life Technologies, Argentina) as previously described. 24 In living cells, MTT is reduced to formazan. Cells (10 4 /well) were seeded in 96-well plates in DMEM/F12 supplemented with 5% fetal bovine serum and then left to adhere 4 h. Subconfluent conditions of about 50-60% were chosen to allow detection of maximal growth.…”

M3 muscarinic acetylcholine receptor in cardiology and oncology

“…Further study is needed to evaluate differences in M 3 -mAChR signaling caused by changes in receptor desensitization, sequestration [35][36][37][38][39], and up-or down-regulation and to elucidate the role of M 3 -mAChR in cardiovascular system as well in inflammation and in cancer while new findings are emerging as regards the use of cardiovascular drugs [38,55,68,69,74,75,[77][78][79]82]. M 3 -mAChR was involved in LPS-induced lung inflammation [84] and fibroblast proliferation [85] by mediating the NF-κB signaling pathway. Evidence suggests that the blockage of M 3 -mAChR exerts antiinflammatory properties [84].…”