RESUMO
Aim To investigate the effects of vitamin C (VC) on glycine receptor (GlyR) subtypes. Methods The HEK-293T cells were transfected with plasmids expressing different subtypes of GlyR. Then the cells were incubated with different concentrations of VC. The EC2 concentration of glycine-activated currents were recorded by patch clamp before or after incubation of VC. The effects of the sodium-dependent vitamin C transporter-type 2 ( SVCT2 ) inhibitor sulfinpyrazone on VC induced potentiation of GlyRs were also examined. The method of amino acid point mutation was used to explore the critical site for interaction between VC and GlyR. Results Ascorbic acid dose-dependently increased the currents mediated by GlyRal and GlyRa3, with a3 subunits being the most sensitive to VC. Ascorbic acid had no significant effect on the current mediated by the al subunit of GlyRs. Cell incubation with sulfinpyrazone did not affect the VC induced potentiation of GlyR function. The mutation of Ser296 at the third transmembrane domain of a3 GlyR significantly reduced the potentiation of VC on GlyR func-tion. Conclusions Ascorbic acid can enhance the function of GlyR al and a3 subunits, but not a2 sub- unit. Such enhancement is not likely to be an effect oc- curing inside cells. The Ser296 of GlyR plays a key role in the VC induced enhancement of GlyR function.
RESUMO
Aim To investigate the effects of morphine preconditioning (MPC) on transient receptor potential vanilloid 1 (TRPV1) channel current in rat dorsal root ganglia (DRG) neurons and the phosphorylation (p) of TRPV1 and extracellular regulated protein kinases (ERK) in PC12 cells that sensitized by nerve growth factor (NGF). Methods DRG neurons isolated from T2-T8 segments of 10 days old SD rat or pheochromo-cytoma (PC12) cells were seeded into 24-well plates or 6-well plates, respectively, and randomly divided into 5 groups:control group (group C), NGF sensiti-zation group (group NGF), and morphine precondi-tioning groups (group MPC 0.3, group MPC 1.0 and group MPC 3.0). DRG neurons were identified by im-munofluorescent method with neuronal specific enolase (NSE). Cells were treated by morphine, NGF and capsaicin to simulate the effects of MPC on DRG neu-rons in T2-T8 segments during myocardial ischemia reperfusion injury (IRI). Afterwards, the inward cur-rent of DRG neurons induced by capsaicin in all groups were detected by whole cell recording; the expression and phosphorylation of TRPV 1 and ERK in PC12 cells were detected by Western blot. Results DRG neu-rons survived and grew nicely,and the staining of neu-ronal specific markers,NSE,was positive. In compar-ison with group C, the inward current of group NGF was enhanced (P <0.05), while MPC inhibited the enhancement (P <0.05). The relative expression of TRPV1,p-TRPV1 and p-ERK in group NGF was up-regulated when compared with group C (P <0.05).Moreover, the up-regulation was also suppressed by MPC (P <0.05). Conclusions MPC inhibits TR-PV1 channel current sensitized by NGF in neurocytes, and the mechanism might be associated with the down-regulation of TRPV1 p-TRPV1 and p-ERK expression.