The Role Of L-type Calcium Channel In The Neuronal Injury Induced By Protein Non-enzymatic Glycoxidation

Protein nonenzymatic glycoxidation is a pathological characteristic in the brain of many neurodegenerative diseases. The mechanisms underlying protein nonenzymatic glycoxidation-mediated neuronal injury are still unclear. We assumed that disturbance in intracellular calcium homeostasis may be involved in this kind of cell death. In the present study, we investigate the effects of high concentration D-glucose, reactive carbonyl compounds (RCO) and advanced glycation end products (AGEs) on neuronal survival, intracellular calcium concentration and L-type calcium channel in cultured hippocampal neurons. The effects of L-type calcium channel agonist and antagonis on protein nonenzymatic glycoxidation-induced changes in neuronal survival, intracellular calcium concentration and L-type calcium channel were also exmined.1. Establishment of a model of protein non-enzymatic glycoxidation-induced neuronal injury in cultured hippocampal neurons(1) High concentration D-glucose induced neuronal injury in cultured hippocampal neuronsTreatment with D -glucose (33.3mM) for 72h reduced neuronal survival rate significantly to 55.9% ± 10.2% (n=48). 3-O-methylglucose, a nonmetabolizable analogue of glucose which can glycosylate proteins, had a similar effect as D-glucoseand reduced neuronal survival to 60.1%+ 12%(n=48). But neurons treated with mannitol at the same concentration did not display any differences in cell survival rate compared with those treated with normal concentration (5mM) glucose. These results indicated that high D-glucose-induced neuronal injury was not due to either glucose metabolism or osmotic effect. Glycoxidation inhibitors aminoguanidine (AMG) and pyridoxamine (PY) remarkably prevented the neuronal death induced by glucose and 3-O-methylglucose in a dose dependent manner. So did the antioxidants N-acetyl-cysteine (NAC) and DL- α thioctic acid (TA). These results demonstrated that high concentration of glucose damaged neurons via protein nonenzymatic glycoxidation modification.(2) Reactive carbonyl compounds and AGEs induced neuronal injury in cultured hippocampal neuronsSurvival rate of cultured neurons treated with 200 μ M, 400 u M or 800 μ M Methylglyoxal were reduced by 41.2%±5.5%, 56.2%±4.5% and 66.5%±5.8% (P (0.01, N=48) after 24h treatment, and 61.1%±7.5% 70.9%±6.5% and 78.1%± 5.5% (P (0.001, N=48) after 48h treatment, respectively.AGEs could also reduce cell survival rate by 23.1%+3.5% after 24h treatment and 45.4%±4.5% after 48h treatment, respectively.AMG, PY, TA and NAC blocked the harmful effect of Methylglyoxal and AGEs on neuronal survival.These results indicated that Mthylglyoxal and AGEs damaged neurons via protein nonenzymatic glycoxidation.From all above, we establish a reliable model of neuronal injury in cultured hippocampal neurons using high concentration D-glucose, intermediate products and end products during protein nonenzymatic glycoxidation modification. 2. Protein non-enzymatic glycoxidation induced a reduction of intracellular freecalcium in cultured hippocampal neuronsThe cultured neurons were treated with D-GLU and 3-o-m-GLU for two days, and then assay of intracellular free calcium was performed by confocal microscopy. 33.3mM D-GLU and 3-O-M-GLU significantly reduced intracellular free calcium concentration from 110.13 ± 12.63nM to 63.03±12.22 nM and 69.4±8.85 nM, respectively. Glycoxidation inhibitors and antioxidants (AMG, PY, NAC and TA) significantly blocked the effects of D-GLU and 3-O-M-GLU, leading to an increase in intracellular free calcium to 88.44 + 8.24 nM,106.29± 13.78 nM, 96.88 + 6.50 nM and 97.13 + 9.14 nM, respectively. The intermediate-product methylglyoxal and end-product AGEs also significantly reduced intracellular free calcium to 56.59 + 4.5746 nM and 62.30 + 4.63 nM. A blocking effect was observed with the glycoxidation inhibitors and antioxidants.These results demonstrated that protein nonenzymatic glycoxidation induced areduction of intracellular free calcium.3. Protein non-enzymatic glycoxidation inhibited L-type calcium channel current incultured hippocampal neurons(1) High concentration D-glucose inhibited L-type calcium currents in culturedhippocampal neurons Whole-cell inward L-type calcium currents were elicited by voltage steps to lOmVfrom a holding potential of -50mV. After 48 hours treatment of D-gluocse or3-O-methylglucose, L-type calcium currents decreased by 48.3%+5.12% and 46.7%±7.52% respectively compared with control group. 1 mM AMG, PY, NAC or TAcould greatly prevent the effect of high concentration D-gluocse and3 -O-methylglucose.The reversal potential and activation threshold of L-type calcium channel wereremained unchanged with treatment of all chemicals used above.(2) Methylglyoxal and AGEs inhibited L-type calcium currents in cultured hippocampal neuronsL-type calcium current significantly decreased after 24 hours of exposure to 400uM Methylglyoxal or 0.35mg/ml AGEs by 68.5%±2.5% and 58.5%±5.74% respectively, when voltage steps to 10 mV from holding potential of-50mV. 1 mM AMG and PY could significantly prevent the effect of Methylglyoxa and AGEs, and so did TA and NAC (P<0.01).These results demonstrated that protein nonenzymatic glycoxidation could reduce L-type calcium currents.4. Involvment of L-type calcium channel in protein nonenzymatic glycoxidation-induced reduction of intracellular calcium concentration in cultured hippocampal neuronsCultured neurons co-treated of Bayk8644, an L-type calcium channel agonist, with D-GLU, 3-o-mGLU, MXG, or AGEs for 24 hour showed an intracellular calcium concentration close to normal level. Exposure to L-type calcium channel blocker Nifedipine itself for 24h decreaed the intracellular calcium concentration but there was no further reduction when co-incubated with D-GLU, 3-O-M-GLU, MXG or AGEs. These results demonstrated that L-type calcium channel contributed to protein nonenzymatic glycoxidation-induced decline in intracellular calcium concentration.5. Contribution of L-type calcium channel to protein nonenzymatic glycoxidation-induced neuronal injury in cultured hippocaml neurons(1) Co-incubation of 500nM Bay k8644 for 24 hour reversed the harmful effects of pretreatment with D-GLU, 3-O-M-GLU, MXG or AGEs on cell survival rate. Exposure to nifedipine itself for 24h decreaed the cell survival rate but there was no further reduction when co-incubated with D-GLU, 3-O-M-GLU, MXG or AGEs. These results demonstrated that L-type calcium channel contributed to proteinnonenzymatic glycoxidation-induced neuronal death.(2) During whole cell L-type calcium currents recording; acute addition of L-type calcium channel agonist Bayk8644 or antagonist nifedipine increased or decreased L-type calcium currents; respectively; in cultured neurons pretreated with D-glucose; 3-O-methylglucose; Methylglyoxal or AGEs for 48h.These results demonstrated that the decrease in L-type calcium currents played an important role in neuronl injury produced by protein nonenzymatic glyoxidation. Taken together; the present study suggests that down-regulation in L-type calcium channel function and declined intracellular free calcium are involved in the hippocampal neuronal injury induced by protein nonenzymatic glycoxidation.