Conclusions In this study, we reported here that ET 1 ET receptor system exerts its effects on COX two gene expression and PGE2 release in mouse bEnd. 3 cells. The Gi and Gq protein coupled ETB receptor, ERK1 two, p38 MAPK, JNK1 two, and NFB cascades cooperatively mediated these effects of ET 1. These findings regarding ET 1 induced COX 2 PGE2 technique imply that ET 1 may well play a critical part in brain in jury, vascular inflammation, and CNS diseases, mediated via MAPK dependent activation of NFB pathway in bEnd. 3 cells. Pharmacological approaches suggest that tar geting COX two PGE2 technique and their upstream signaling components need to yield valuable therapeutic targets for brain injury and inflammatory diseases. Procedures Materials Dulbeccos modified Eagles medium F 12 medium, fetal bovine serum, and TRIzol have been from Invitrogen.
Hybond C membrane and enhanced chemiluminescence Western blot selleck chemicals detection system had been from GE Healthcare Biosciences. Anti COX 2 monoclonal anti body was from BD Transduction Laboratories. Phospho ERK1 two, phospho p38, phospho JNK1 2 antibody kits were from Cell Signaling. p65, p42, p38, and JNK1 antibodies have been from Santa Cruz. Anti glyceraldehyde 3 phosphate dehydrogenase antibody was from Biogenesis. BQ 123, BQ 788, GP antagonist two, GP antagonist 2A, U0126, SB202190, SP600125, and Bay11 7082 have been from Biomol. Bicinchoninic acid protein assay reagent was from Pierce. ET 1, enzymes, along with other chemical compounds were from Sigma. Mouse brain microvascular endothelial cell culture Mouse brain microvascular endothelial cells were bought from Bioresource Collection and Re search Centre and grew in DMEM F 12 containing 10% FBS and antibiotics at 37 C inside a humidified 5% CO2 atmos phere.
The cell line is acquired from mouse BALB c strain brain cerebral cortex endothelial polyoma selleckchem middle T antigen transformed, which was performed STR PCR profile at BCRC. Each of the experiments were performed applying this cell line and approved by the ethic approval of Chang Gung University. Confluencent cells have been released with 0. 05% trypsin 0. 53 mM EDTA for 5 min at 37 C. The cell suspension was plated onto six properly culture plates or 10 cm culture dishes for the measurement of pro tein or RNA expression, respectively. Culture medium was changed just after 24 h and after that each 3 days. Experi ments had been performed with cells from passages 5 to 13.
Preparation of cell extracts and Western blot analysis Growth arrested cells were incubated with ET 1 at 37 C for several time intervals. The cells were washed with ice cold phosphate buffered saline, scraped, and collected by centrifugation at 45,000 ? g for 1 h at four C to yield the whole cell extract, as previously described. Samples had been analyzed by Western blot, transferred to nitrocellulose membrane, and after that incubated over evening utilizing an anti COX two, phospho ERK1 two, phospho p38 MAPK, phospho JNK1 2, p42, p38, JNK1, p65, or GAPDH antibody.