effects of midazolam and ketamine on cytoskeleton of hepatocytes

• Main Authors: Lin,Yu-Hua, 林鈺樺
• Other Authors: Chen, Ruei-Ming
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/65057757723144698933

碩士 === 臺北醫學大學 === 醫學研究所 === 93 === Midazolam, an imidazobenzodiazepine derivative, is utilized as an intravenous anesthetic agent. The hypnotic effect of midazolam probably is related to gamma-aminobutyric acid accumulation. Ketamine is an intravenous dissociative anesthetic agent, whose mechanism of action might be an N-methyl-D-aspartate receptor antagonist. Both drugs are metabolized by cytochrome P450-dependent monooxygenase system, mostly 3A4 isoform, in the liver. Cytoskeleton is the major organelles in the cytoplasm, which is important for the architecture, motility, metabolism, and intracellular signal transduction of the cell. There are three types of protein filaments in the cytoskeleton, including microfilaments, microtubules, and intermediate filaments. Cytoskeleton is closely connected with plasma membrane; therefore, it is of interest when considering the effects of lipid-soluble anesthetics. Evidence showed that filament-disrupting agents, such as cytochalasin, alter cellular response, and certain general anesthetics modulated cytoskeletal organization. This study was aimed to elucidate the effects of midazolam and ketamine on cytoskeleton of hepatic cells, using human HepG2 cells as the experimental model. The results demonstrated that, in the clinically relevant concentration, midazolam（0.5 μM）and ketamine（100 μM）did not affect viability of cells up to 24 hours. Cells were stained with TRITC-phalloidin that specifically binds filamentous actin, and observed using fluorescence microscopy and laser scanning confocal microscopy. Exposures to midazolam or ketamine for 24 hours changed microfilament distribution and reduced microfilament contents within cells. Reverse transcriptase-polymerase chain reaction assay was carried out to determine the effects of midazolam and ketamine on the synthesis of actin. Midazolam induced α-actin mRNA synthesis without affecting the transcription of β-actin. Neither β-actin nor α-actin mRNA production was affected by ketamine administration. Immunocytochemistry analysis was carried out using anti-α-tubulin-FITC antibodies to determine the effects of midazolam and ketamine on microtubule cytoskeleton. Microtubule structure was disorganized after exposure to either midazolam or ketamine for 6 and 24 hours. Erythromycin N-demethylation and pentoxyresorufin O-dealkylase assays were carried out to determine the effects of midazolam and ketamine on enzyme activities of cytochrome P450 3A4 and 2Bs. Neither 3A4 nor 2Bs activity was affected by midazolam or ketamine. Reverse chain-polymerase chain reaction was performed to analyze the effects of midazolam and ketamine on the synthesis of cytochrome P450 mRNAs. Midazolam did not affect the production of cytochrome P450 3A4 and 2B6 mRNA. Ketamine inhibited cytochrome P450 2B6 mRNA synthesis after exposed for 6 and 24 hours. Our results imply that hepatic cytoskeleton might be modulated by midazolam and ketamine. Changes in distribution of microfilaments and disorganization of microtubules in hepatocytes might affect normal hepatic function, e.g. cytochrome P450. Further studies are necessary to clarify the consequence and clinical importance.