ABSTRACT
OBJECTIVE To investigate the inhibitory effect and mechanism of lead(Pb2+)on γ-amino-butyric acid(GABA)A receptor-mediated currents(IGABA)and GABAergic synaptic transmission in rat cortical neurons.METHODS ①The cortical neurons from 0 d Sprague Dawley(SD)rats were cultured for experiments.The cultured cells(7-14 d)were recorded using the patch-clamp technique to analyze the effects of Pb2+ at different concentrations(1,5,10,50 and 100 μmol·L-1)on IGABA induced by GABA 100 μmol·L-1.② The effects of Pb2+ 50 μmol·L-1 on IGABA induced by GABA at different concentrations(1,10,50,100,500 and 100 μmol·L-1)were detected.③Brain slices(350 μm)were prepared from SD rats(15-19 d).The spontaneous inhibitory post-synaptic currents(sIPSCs),miniature inhibitory post-synaptic currents(mIPSCs)and current injection-induced action potential(AP)were recorded to detect the effects of Pb2+ 10 μmol·L-1 on the amplitude and frequency of sIPSCs and mIPSCs,and the frequency of AP.RESULTS ①Pb2+ inhibited IGABA in a concentration-dependent manner,and IC50 was(68±20)μmol·L-1.②Pb2+ also suppressed the maximum current induced by GABA(P<0.01),with a significant increase of the GABA′s EC50 from(20±6)μmol·L-1 to(87±39)μmol·L-1,indicating that Pb2+ might inhibit IGABA in a non-competitive mechanism.③Pb2+ 10 μmol·L-1 inhibited the frequency(P<0.01)rather than the ampli-tude of sIPSCs reversibly,but had no effect on eigher the frequency or amplitude of mIPSCs.In addi-tion,Pb2+ decreased the frequency of evoked AP by current injection(P<0.01)and reduced the overall excitability of rat cortical neurons.CONCLUSION Pb2+ can significantly inhibit IGABA in primary cultured neurons.In the brain slice experiment,Pb2+ may affect sIPSCs frequency by inhibiting the AP of cortical neurons,suggesting that there are different intrinsic mechanisms through which Pb2+ inhibits both IGABA in primary cultured neurons and the frequency of sIPSCs in brain slice neurons,which points to the complexity of the mechanism of Pb2+ poisoning.
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Objective To clarify the protective effect of allopregnanolone (APα) on cell line SH-SY5Y damaged by 6-hydroxydopamine (6-OHDA) and its possible molecular mechanism. Methods 6-OHDA, APα, γ-aminobutyric acid A receptor (GABAAR) antagonist, voltage-gated L-type Ca2
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Objective To evaluate the effect of mechanical stretch preconditioning on pathological stretch-induced activation of γ-aminobutyric acid (GABA) signaling pathway in human type Ⅱ alveolar epithelial cells (AEC Ⅱ).Methods AEC Ⅱ cell line (A549 cells) culturedin vitro were divided into control group (group C), pathological stretch group (group P1) and mechanical stretch preconditioning group (group P2). In group C, A549 cells were cultured routinely. In group P1, A549 cells were exposed to 20% cyclic stretch for 6 hours. In group P2, A549 cells were exposed to 5% cyclic stretch for 60 minutes, and then exposed to 20% cyclic stretch for 6 hours. The cells were harvested for determination of the cell viability by methyl thiazolyl tetrazolium assay, lactate dehydrogeuase (LDH) release was determined by colorimetric method, the levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA), the mRNA expressions of IL-1β, IL-6 and TNF-α were determined by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expressions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid A receptor (GABAAR) were determined by Western Blot.Results Compared with group C, the cell viability of group P1 was significantlydecreased (A value: 0.196± 0.071 vs. 0.886±0.107), the release rate of LDH was significantly increased [(12.3±2.4)% vs. (1.9±0.5)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly increased [IL-1β (ng/L): 138.6±19.7 vs. 32.7±7.4, IL-6 (ng/L): 196.5±31.7 vs. 55.4±13.8, TNF-α (ng/L): 111.3±21.8 vs. 20.8±7.6; IL-1β mRNA (2-ΔΔCT): 2.79±0.44 vs. 0.83±0.12, IL-6 mRNA (2-ΔΔCT): 1.99±0.25 vs. 0.56±0.11, TNF-α mRNA (2-ΔΔCT): 2.54±0.37 vs. 0.72±0.09]; the protein expressions of GAD and GABAAR were significantly decreased [GAD (gray value): 0.38±0.12 vs. 1.75±0.45, GABAAR (gray value): 0.29±0.09 vs. 1.68±0.39; allP < 0.05]. Compared with group P1, the cell viability of group P2 was significantly increased (A value: 0.523±0.132 vs. 0.196±0.071),the release rate of LDH was significantly decreased [(6.9±1.7)% vs. (12.3±2.4)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly decreased [IL-1β (ng/L): 79.2±11.6 vs. 138.6±19.7, IL-6 (ng/L): 89.6±15.6 vs. 196.5±31.7, TNF-α (ng/L): 55.9±11.4 vs. 111.3±21.8; IL-1β mRNA (2-ΔΔCT): 1.92±0.36 vs. 2.79±0.44, IL-6 mRNA (2-ΔΔCT): 1.09±0.18 vs. 1.99±0.25, TNF-α mRNA (2-ΔΔCT): 1.77±0.25 vs. 2.54±0.37]; the protein expressions of GAD and GABAAR were significantly increased [GAD (gray value): 1.26±0.33 vs. 0.38±0.12, GABAAR (gray value): 1.04±0.15 vs. 0.29±0.09; allP < 0.05]. Conclusion The mechanism by which mechanical stretch preconditioning attenuates pathological stretch-induced injury in human AECⅡ is related to the activation of GABA signaling pathway.