ABSTRACT
OBJECTIVE: To investigate whether erythropoietin (EPO) protects neuron against ketamine induced injuries. METHODS: Neurons were obtained from SD rat brain, cultured, and treated with ketamine of the concentrations of 0.1, 1, 10, and 30 micromol/L respectively. Neurons not treated by any agent were used as control group. Another neurons were divided into 3 groups undergoing the treatment of ketamine of the terminal concentration of 10 micromol/L, EPO + ketamine group undergoing the treatment of 10 micromol/L ketamine and EPO of the terminal concentrations of 0.3, 1, 3, and 10 U/ml, and ketamine + EPO + LY294002 group undergoing the treatment of 10 micromol/L ketamine, 1 U/ml EPO, and 10 micromol/L LY294002, a P13k inhibitor. Twenty-four hours after the co-inoculation the survival rates of the neurons were detected by MTT method. The apoptotic rate was detected by TUNEL assay. The neuron vitality was measured by MTT assay. Apoptotic neurons were measured by TUNEL assay. The activity of caspase-3 was detected with the caspase-3 fluorometric assay kit. The level of pAkt protein was analyzed by Western blotting. RESULTS: The survival rates of the neurons exposed to ketamine of the concentrations of 1, 10, and 30 micromol/L were (91 +/- 5)%, (42 +/- 6)%, and (23 +/- 7)% respectively, significantly lower than that of the control group (P < 0.05 or P < 0.01). The survival rates of the neurons treated by 10 micromol/L ketamine and EPO of the concentrations of 0.3, 1, 3, and 10 U/ml were (73 +/- 6)%, (86 +/- 9)%, (78 +/- 8)%, and (71 +/- 10)% respectively, all significantly high than that of the 10 micromol/L ketamine group (P < 0.05 or P < 0.01). The number of apoptotic neurons of the 10 micromol/L ketamine group was significantly higher than that pf the control group, the number of apoptotic neurons of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly lower than that of the 10 micromol/L ketamine, and the number of apoptotic neurons of the ketamine + EPO + LY294002 group was (130 +/- 30)%, remarkably lower than that of the ketamine group. (P < 0.01), and the relative activity of caspase-3 of the 10 micromol/L ketamine group was (280 +/- 60)%, significantly higher than that of the control group, (P < 0.01). The relative activity of caspase-3 of the ketamine + EPO + LY294002 group was (220 +/- 34)%, significantly higher than that of the ketamine + EPO (P < 0.01). The pAkt protein level of the 10 micromol/L ketamine group was significantly lower than that of the control group (P < 0.05), the pAkt protein level of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly higher than that of the 10 micromol/L ketamine group (P < 0.01), and the pAkt protein level of the ketamine + EPO + LY294002 group was significantly lower than that of the ketamine + 10 U/ml EPO group (P < 0.01). CONCLUSION: EPO affords significant neuroprotection against ketamine induced injury in neurons via PI3K/Akt-mediated signaling pathway.
Subject(s)
Erythropoietin/pharmacology , Ketamine/toxicity , Neurons/drug effects , Animals , Apoptosis , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Neurons/cytology , Neurons/metabolism , Phosphorylase b/biosynthesis , Rats , Rats, Sprague-Dawley , Recombinant ProteinsABSTRACT
The effect of specific ligands on the initial rate of muscle glycogen phosphorylase b digestion by trypsin has been studied. The kinetics of tryptic proteolysis were followed by measuring the decrease in phosphorylase b fluorescence intensity at 335 nm (excitation at 290 nm). The kinetic curves were linear at least in the region 0-400 s (0.02 M HEPES, pH 6.8; 37 degrees C). An allosteric activator (AMP) and allosteric inhibitors (flavins) protected the enzyme from tryptic digestion when trypsin was added to the enzyme preincubated with the ligand. Differences were found between the kinetic curves of trypsinolysis initiated by addition of the trypsin-ligand mixture to phosphorylase b an by addition of trypsin to the enzyme preincubated with the ligand for 10 min. It is concluded that the specific ligands under study (AMP, flavins, and the substrate--glucose 1-phosphate) induce relatively slow conformational changes in the phosphorylase b molecule with the half-conversion time of several minutes.
Subject(s)
Muscles/enzymology , Phosphorylase b/chemistry , Protein Conformation , Adenosine Monophosphate/pharmacology , Allosteric Regulation , Animals , Enzyme Induction , Flavin Mononucleotide/pharmacology , Flavin-Adenine Dinucleotide/pharmacology , Hydrolysis , Kinetics , Ligands , Phosphorylase b/biosynthesis , Phosphorylase b/metabolism , Rabbits , Spectrometry, Fluorescence , Trypsin/metabolismABSTRACT
Evidence is presented that the sarcoplasmic reticulum (SR)-glycogenolytic complex isolated from fast twitch skeletal muscle is a highly specific, functionally defined compartment for phosphorylase regulation. The addition of ATP alone results in prompt phosphorylase activation which demonstrates calcium dependence similar to the calcium-magnesium ATPase that catalyzes SR calcium transport suggesting that these two calcium-requiring -ystems might interact within the complex. Lowering extravesicular calcium concentration by transport of calcium into the SR lumen resulted in inactivation of phosphorylase a. This effect could be prevented by the addition of the calcium ionophore X537A which inhibits SR calcium sequestration or a calcium EGTA buffer which maintains free calcium. It was mimicked by EGTA addition. Since exogenous phosphorylase b and phosphorylase a were not activated or inactivated, respectively, by the endogenous activating enzymes or phosphatase in the SR-glycogenolytic complex, these regulatory enzymes may be compartmented. In addition, endogenous phosphorylase could be uncoupled from its activating enzymes by amylase treatment. These results suggest that the SR-glycogenolytic complex in fast twitch skeletal muscle is a compartmented system for phosphorylase activation controlled by SR calcium flux, a feature in contrast to the cardiac complex (Entman, M.L., Kaniike, K., Goldstein, M.A., Nelson, T.E., Bornet, E.P., Futch, T.W., and Schwartz, A. (1976) J. Biol. Chem. 251, 3140-3146). We suggest that the complex is the in vitro counterpart of the well documented rapid burst of glycogenolysis which ensures with the onset of contraction.