ABSTRACT
OBJECTIVE Investigate the effects of diazepam (DZP) on γ2 subunit containing GABA type A receptor (GABAAR) trafficking. METHODS Immunofluorescence microscopy measured surface GABAARs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol · L- 1 DZP. Biochemical studies of mice injected with 10 mg·kg-1 DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post- injection. Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L- 1 DZP exposure. A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein (γ2pHFAP) measured lysosomal targeting of γ2 containing GABAARs. RFP-gephyrin and γ2pHFAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching (FRAP). RESULTS Extrasynaptic levels of γ2 GABAARs decreased by 12.2%, while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control (P<0.05). Dendritic levels of gephyrin were also reduced to 74.1% of control, while S270 phosphorylation was elevated by 25.2% (P<0.05; P<0.01). Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels, respectively (P<0.05; P<0.01). 12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control (P<0.05). Internalized γ2pHFAP GABAARs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control. Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP. CONCLUSION DZP treatment decreases γ2 GABAAR levels and gephyrin scaffolding function after one day of exposure, which may contribute to the formation of DZP tolerance.
ABSTRACT
To evaluate the free radical generation and antioxidant enzymes status in murine peritoneal macrophage during in vitro amikacin resistant Pseudomonas aeruginosa (ARPA) treatment with different time interval. Methods: Peritoneal macrophages were treated with 1×108 CFU/mL ARPA cell suspension in vitro for different time interval (1, 2, 3, 6, 12, and 24 h) and super oxide anion generation, NO generation, reduced glutathione level and antioxidant enzymes status were analyzed. Results: Super oxide anion generation and NO generation got peak at 12 h, indicating maximal free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during ARPA transfection. Reduced glutathione level and antioxidant enzymes status were decreased significantly (P<0.05) with increasing time of ARPA transfection. All the changes in peritoneal macrophages after 12 h in vitro ARPA transfection had significant difference (P<0.05). Conclusions: From this study, it may be summarized that in vitro ARPA infection not only generates excess free radical but also affects the antioxidant system and glutathione cycle in murine peritoneal macrophage.