OBJECTIVETo investigate the transcriptional regulation of pacemaker channel I(f) mediated by vasoactive peptide endothelin-1 (ET-1) in neonatal rat ventricular myocytes and its mechanism.

METHODSNeonatal rat ventricular myocytes were enzymatically isolated. I(f) current was recorded using the whole-cell patch-clamp technique. The expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) isoforms HCN2 and HCN4 were measured by quantitative RT-PCR.

RESULTSET-1 increased the expression of HCN2 and HCN4 mRNA in a dose- and time-dependent manner. These effects were blocked by specific ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. The effects of ET-1 on HCN2 and HCN4 mRNA expression were not affected by the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-203580).

CONCLUSIONThese findings indicate that ET-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes via ETA receptor through a p38 MAPK-independent signaling pathway, which might be linked to the intrinsic arrhythmogenic potential of ET-1.

Animals ; Animals, Newborn ; Cyclic Nucleotide-Gated Cation Channels ; drug effects ; Endothelin-1 ; metabolism ; Imidazoles ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective : To investigate the effects of hederagenin (HDG) on proliferation , migration , invasion and apoptosis of glioblastoma (GBM) cells and involved mechanism. Methods : Human GBM cell lines U87 , U251 and human brain glial cell line (HEB) were selected as the study subjects , and HDG 0 μmol/L ( or 0 mg/kg) was used as the control group. MTT , EdU staining and cell plate cloning were used to detect the effect of HDG on the proliferation of GBM cells. Trypan blue staining was used to detect GBM cell death affected by HDG. The effects of HDG on migration and invasion of GBM cells were detected by cell scratch and Transwell assay. To analyze the effects of HDG on apoptosis of GBM cells , apoptosis⁃related proteins Bcl⁃2 , Bax , p53 and cleaved caspase⁃3 were detected by Western blot. Mitochondrial potential change was detected by JC⁃10 staining , and apoptotic cell count was displayed by Annexin V ⁃FITC staining. The effect of HDG on tumor bearing in GBM was analyzed by xeno transplantation in BALB/C mice. Results : Compared with the control group (HDG 0 μmol/L) , HDG significantly inhibited the proliferation , migration and invasion of U87 and U251 cells , and they were dependent on the use dose of HDG. Trypan blue staining showed that HDG obviously increased death number of GBM cells. The mitochondrial potential of GBM cells was remarkedly decreased , the number of apoptotic GBM cells obviously increased , the expressions of apoptosis⁃related proteins p53 , Bax , cleaved⁃caspase3 were up⁃regulated and Bcl⁃2 was down⁃regulated by HDG in U87 and U251 cells. HDG significantly inhibited the size of subcutaneous GBM , the Ki67 positive rate of GBM cells and caused a large number of GBM cells to die in BALB/C mice. HDG had no obvious toxic effect on human HEB cells and the liver of tumor⁃bearing mice. Conclusion HDG can significantly inhibit the proliferation , migration and invasion of GBM cells and induce the apoptosis of them. The mechanism of HDG induced apoptosis of GBM cells may be through mitochondrial damage and regulation of p53 and Bcl⁃2/Bax expression.