The Influence Of Vascular Endothelial Growth Factor On Calcium Channel Of Neurons

Objective: In: the nervous system,calcium as an important second messenger,regulating many important functions,such as neuronal excitability and synaptic plasticity.It can lead to neuronal injury or death when calcium ions in the intracellular accumulation,namely calcium overload occurs.When intracellular calcium overload occurs,multiple Ca²⁺ dependent signal transduction pathway will be activated,and thus stimulate a variety of complex time and space cascade reactions.The mechanism mainly include:oxidation of free radicals increased and increased NO,increased destruction of mitochondria and cytoskeleton destruction.It directly exerts its neuroprotective function through the inhibition of cell death?PCD?and the inhibition of apoptosis and the stimulation of the nerve.Recent studies have shown that vascular endothelial growth factor?The vascular endothelial growth factor,VEGF?,a kind of hypoxia induced proteins,not only has the angiogenesis and vascular permeability-enhancing properties but also has neurotrophic and neuroprotective as well as angiogenic properties.VEGF is also a mediator in many indirect neural protective processes,including angiogenesis,increased the permeability of blood brain barrier to glucose and the antioxidant activation.VEGF is also expressed on the surface of neurons and protect neurons from death under conditions such as hypoxia or lack of glucose by binding to specific receptors.Recent studies have indicated that VEGF can also inhibit delayed outward integration of potassium currents in acutely isolated hippocampal neurons by potassium channel Kv1.2 protein tyrosine phosphorylation.There are also literature on hippocampal slices which reports that VEGF can inhibit the postsynaptic potential induced by electrical stimulation of excitatory synaptic pathways.However,in the physiology and pathology of the nervous system changes,the effect of VEGF on neuronsvoltage dependent calcium channel remains unknown.This experiment using the electrophysiology research and laser confocal calcium imaging techniques to explore whether VEGF exert its nerve protective effect by increasing the voltage dependent calcium channel activation of neurons and decreasing calcium ion into the nerve cells through the voltage dependent calcium channel to inhibit the intracellular calcium overload.Methods:1 Primary hippocampal neurons was vaccinated in a small Petri dishes in 0.5×10⁶ density and cultured for 7 days normally.The control group was added VEGF?100ng / ml?and incubating for 24 hours.The control group only was added an equal volume of medium incubating for 24 hours.The whole cell patch clamp recording was performed under the same conditions,to observe the effect of VEGF on whole cell calcium currents in cultured hippocampal neurons.2 We labeled cultured Primary cultured hippocampal neurons intracellular Ca²⁺ with Fluo-4/AM,using confocal laser scanning microscopy?CLSM?continuously scan the intracellular calcium fluorescence intensity before and after the high potassium?KCl?of stimulation,and indirectly reflected the intracellular free calcium ion concentration changes.Then,we can detect the effect of VEGF pretreatment on the intracellular calcium overload induced by potassium.Results:1 The whole cell patch clamp recording was performed under the same conditions.Get a small dish and clean it with the standard extracellular solution and add electrode extracellular solution which to record calcium current.The whole-cell inward Ca²⁺ current was evoked by depolarization from -80 to +40mV at a holding potential of -80mV with 300 ms wave width and recorded under a condition of Ba²⁺ instead of Ca²⁺ in bath solution to reduce of Ca²⁺ current rundown.And then we recorded the whole cell calcium channel current of two groups of cells and transformed into the currentdensity.The depolarization potential of control group and VEGF pretreatment group respectively are -10mV and +20mV when reached peak value of current density.The maximum current density was not statistically significant difference by using statistical t-test analysis?P>0.05?.This indicate that VEGF did not affect the maximum value of the whole cell calcium currents.2 The control group and VEGF pre treatment group current density curve fitted with Boltzmann equation.So we can get it that the control group and VEGF pretreatment group half activation voltage are-4.0mV and -34.2mV.By the statistical analysis,the two group have significant differences.This showed that VEGF pretreatment changed the activation characteristics of cultured hippocampal neurons,increased the activation voltage,making it more difficult to activate.3 In control group and VEGF pretreatment group,the calcium concentration in the cells increased rapidly after the addition of high potassium solution,and then decreased slowly.The increase of intracellular calcium concentration in VEGF pretreatment group was significantly lower than that in control group.This shows that Vascular endothelial growth factor?VEGF?pretreatment inhibited the increase in intracellular calcium concentr-ation induced by the high potassium?According to the Nernst equation the concentration is equivalently Depolarization potential of +20mV?.Group statistical analysis there was a significant difference of calcium ion fluorescence density between the control group and VEGF pretreatment?control group,706.88+56.37%,n=11;VEGF pretreatment group,450.55 + 31.05%,n=9??P < 0.05?,indicating that VEGF can inhibit the intracellular calcium overload.Conclusions:VEGF can decrease calcium ion into the nerve cells through the voltage dependent calcium channel and inhibit the intracellular calcium overload by increasing the voltage dependent calcium channel activation of neurons.