Research About The Relationship Between Hypoxia Injury And Sodium Pump Function In Cortical Neurons

Cerebral ischemia results in severe cell degeneration and consequently loss of brain functions. Now, it is widely accepted that ischemic neuronal cell death results from excessive intracellular accumulation of Ca2+ caused by the massive release of glutamate during the insult.The Na pump, or Na+, K+-ATPase, is a membrane-bound protein. By utilizing the energy from the hydrolysis of one molecule of ATP, it translocates three Na+ out of the cell and two K+ into the cell. The electrochemical gradient the Na pump generates is critical in maintaining the osmotic balance of the cell and the resting membrane potential of most tissues. Thus the Na pump is essential in the maintenance of Na+, body fluid and electrolyte homeostasis.When the supply of oxygen or blood flow to the mammalian brain decreases to critical levels, energy failure occurs, with a decline in ATP by as much as 90% in within 5 min. When 50–65% of the ATP is lost, Na pump activity is inhibited and depolarization of the membrane voltage and subsequent uptake of sodium and water occurs. Depolarization causes Ca2+ influx through voltage-gated Ca2+ channels. The collapse of Na+ gradient causes the sodium-glutamate cotransporters to eject glutamate into the extracellular space. Glutamate triggers vigorous activation of glutamate receptors and the Ca2+-dependent cell injury.Obviously, Na pump plays an important role in the occurrence and development of neural damage after cerebral ischemia. Previous studies about the pump are focused on cadiomycyte, kidney, musculi skeleti, and vascular smooth muscle. Research on it in neurons, especially about its role in cerebral ischemic injury, has not yet been conducted extensively. The present study, carried out in cortical neurons (pyramidal neuron of layer V and VI) which are sensitive to hypoxia, is to explore the changes in the electrophysiological characteristics of Na pump in hypoxic injury of cortical neurons and underlying mechanism.I Electrophysiolofical characteristics of Na pump in neurons from cortical slices.Objectives: This part of investigation was undertaken to characterize the Na pump current in neurons from cortical slices.Methods: The young SD rats (11–14 d postnatal) were used to prepare the cortical slices. Animals were deeply anesthetized and decapitated. The brains were quickly removed and submerged in ice-cold ACSF. Sections (300μm thick) of frontal cortex were cut, immersed in ACSF aerated with a mixture of 95% O2/5% CO2. And then the slices were perfused with ACSF gassed with 95% O2/5% CO2 continually after putting them into filling trough and under the fluid. We moved the electrode above neuron by infrared differential interference contrast (DIC) optics, and then made a tight seal and broke the membrane by giving negative pressure. As pipette solution was dialyzing into the cell and membrane current reached a steady-state level at a holding potential of -60 mV, at first we identified that the recording cell was a neurons by membrane depolarization (-60mV—0mV—-60mV) evoking Na current, and then the bath solution was switched from normal ACSF to the ACSF contained Ouabain (Oua, 10-12~10-3 mol/L), which resulted in a inward shift of the membrane current. The Na pump current was determined as the difference in holding current levels in the absence and presence of Oua, and was identified as Oua-sensitive current by the way that the amplitude evoked by 1 mmol/L Oua equaled to that induced by free-K+ extracellular fluid. Then Oua-sensitive currents were measured as the concentration of Oua ([Oua]) ranged from 10-12 to 10-3 mol/L and a Oua dose-response curve was constructed. The Oua-sensitive currents (?Ip, stimulation, or inhibition) were normalized to the maximal value of ?Ip obtained in the same cell by total pump inhibition on application of 10-3 mol/L Oua and . Na pump current was also identified as the voltage-sensitive current and current-voltage (I-V) relationship was observed by square voltage steps to membrane potentials between +40 and -90 mV. The whole experiment was executed under the conditions of room temperature 23±2℃.Results: All cells in the experiments were pyramidal neurons because of Na+ current and larger volume and Oua-sensitive current represented Na pump current because the amplitude evoked by 1 mmol/L Oua equaled to that induced by free-K+ extracellular fluid.Perfusion of the ACSF contained different concentration of Oua evoked an inward shift of the holding current at -60mV in a concentration-dependent manner. The Na pump current was stimulated or inhibited by concentrations of Oua (10-12~10-3 mol/L), which were divided into three models, stimulation of Ip, inhibition of Ip, and no change of Ip, based on response of Na pump in neurons to low concentration Oua (10-8 mol/L).In the ?Ip-[Oua] relation curve for the stimulation of Ip (in 29 from 96 neurons), the ?Ip values produced by each concentration of Oua from 10-12 to 10-3 mol/L were 0.027±0.114, 0.032±0.082, 0.050±0.213, 0.172±0.226, 0.125±0.152, -0.035±0.036, -0.146±0.124, -0.226±0.161, -0.421±0.076, -0.638±0.138 and -1, respectively. Oua from 10-12 to 10-8 mol/L excited Na pump current and the excitory extend reached its peak at 10-9 mol/L, but concentration-dependent inhibition of Na pump current were recorded by using Oua from 10-7 to 10-3 mol/L. The curve was well fitted using a three-binding site model and the dissociation constants for high-affinity stimulatory binding site, high-affinity inhibitive binding site, and low-affinity inhibitive binding site were 0.31 nmol/L, 41.27 nmol/L, and 152.48μmol/L.In the ?Ip-[Oua] relation curve for the inhibition of Ip (in 61 from 96 neurons), all the ?Ip values on the curve (Oua from 10-12 to 10-3 mol/L) were -0.011±0.072, -0.028±0.071, -0.057±0.079, -0.093±0.063, -0.115±0.069, -0.124±0.067, -0.157±0.094, -0.281±0.144, -0.421±0.076, -0.690±0.112 and -1.0, respectively. Both Oua had inhibitory effects on Na pump current, but this curve displayed nearly plateau level at concentrations of 10-8 and 10-7 mol/L. The curve was well fitted using a two-binding site model and the dissociation constants for high-affinity inhibitive binding site and low-affinity inhibitive binding site were 71.12μmol/L and 176.51μmol/L.In the ?Ip-[Oua] relation curve for the no change of Ip (in 6 from 96 neurons), Oua from 10-12 to 10-7 mol/L had no effect on Na pump current and concentration-dependent inhibition of this current was recorded significantly by Oua from 10-6 to 10-3 mol/L. The curve was well fitted using one-binding site model with dissociation constant for low-inhibitive-affinity site of 149μmol/L.The high-affinity pumps generated 14.59% of the total Na pump current and very likely included theα2 andα3 isoform. The low-affinity pump corresponding to 85.41% of the total Na pump current attributed toα1 isoform. Additionally, the Na pump current exhibited a voltage-dependence; its I-V curve displayed a positive slope at potentials between -90 ~ +40 mV and the reversal potential was near -20 mV.Conclusion: Cortical pyramidal neurons express two functionally distinct pump as expected for high-affinity pump (α2 andα3 isoform) and low-affinity pump (α1 isoform). Na pump current is not only Oua-sensitive, but also voltage-dependent.II Effects of hypoxia on Na pump in cortical neuronsObjection: This part investigation was designed to examine how hypoxia affected Na pump activity and itsα-isoform expression in cortical neurons.Methods: Neurons were exposed to oxygen glucose deprivation by①neural culture under N2 and non-glycose conditions,②or cortical slices perfusion with free-glucose and low-oxygen ACSF. In order to determine whether the two hypoxic method were successfully, we examined the levels of lactate dehydrogenase (LDH) in culture medium at 0, 2, 4, 8 and 12 hour after hypoxia. The intracellular Ca2+ concentration ([Ca2+]i) (fluorescent intensity of fluo-3) was detected in cultured cortical neurons suffered from 0, 0.5, 1, 2, 4 and 8 hour hypoxia using confocal microscope, and TTC staining was employed to evaluate activity of cortical slices after perfusing free-glucose and low-oxygen ACSF at 0, 5, 10, 15, 30, 45 and 60 min. Change of Na pump activity induced by hypoxia in cultured cortical neurons was determined at 0, 2, 4 and 8 hours by inorganic phosphate spectrophotometry, and in cortical slices at 0, 15, 30 and 60 min by double enzymic method. Changes of Na pump current induced by hypoxia in pyramidal neurons from cortical slices were also examined at 0, 2, 4, 6, 8 or 10 min after hypoxia. After Oua of 1mmol/L perfusion, cortical slices were received a washout with normal ACSF and free-glucose and low-oxygen ACSF, to understand the recover velocity of Na pump function.And the expressions of mRNA and protein of Na pumpα1 andα3 isofrom were determined by RT-PCR and Western blot to evaluate effects of hypoxia on Na pump at 0, 4, 8 and 12 hour after hypoxia in cultured cortical neurons, and at 0, 5, 10, 15, 30 and 60 min after hypoxia in cortical slices.Results: LDH in medium of cultured cortical neurons increased from 324±16 to 417±50 U/L (p<0.05), and elevated further as prolonging hypoxic time, suggesting a hypoxic injury. Intracellular Ca2+ overload is another indicator to show cell damage, [Ca2+]i in cultured cortical neurons raised and reached its peak 1.71±0.46 within 1 hour, and maintained the maxiamal values for 2~8 hour hypoxia. TTC staining showed that the activity of cortical slices reduced to 0.292±0.019 (P<0.01) after perfusing free glucose and low oxygen ASCF for 5min, the lower level of the activity was kept until 15 min, and then dropped significantly (0.257±0.012, P<0.01).The activity of pump increased from 1626±122 to 4114±472μmol/NADH/mg·protein/h (p<0.01) after hypoxic culture for 2 hours, and then with prolonged hypoxic time, gradually decreased to 864±318μmol/NADH/mg·protein/h (p<0.01) at 4-hour after hypoxia and to 497±86μmol/NADH/mg·protein/h (p<0.05) at 8-hour after hypoxia. In additionally, this pump activity of cortical slices increased from 169±32 to 572±28μmol/mg protein/h (P<0.05) at 30 min after hypoxia, and then decreased at 60 min after hypoxia (243±72μmol/mg protein/h, P<0.05).Na pump current of cortical slices after hypoxic exposure increased and then decreased. The pump current was significantly lower at 4 min after hypoxia compared with control (0.265±0.068 vs 0.160±0.046 pA/pF,P<0.01), but it enhanced to 0.243±0.054 pA/pF (P<0.05) at 20 min after hypoxia. After holding current was inwardly shifted by 1 mmol/L Oua, the perfusion of free-glucose and low oxygen ACSF did not eliminate the inhibition of sodium pump function to recover the holding current to the original level before giving Oua, suggesting that hypoxia inhibited Na pump function.Hypoxic culture caused an increase in the expression ofα1 isoform mRNA in cultured cortical neurons, the mRNA levels were 0.822±0.050, 0.873±0.096, 0.960±0.089, 0.938±0.080 and 0.829±0.098, respectively, at 0, 2, 4, 8 and 12 hour after hypoxia, and there were significant differences (p<0.01) at 4 hour and 8 hour after hypoxic exposure. In contrast hypoxic culture caused a decrease in the expression ofα3 isoform mRNA, the mRNA levels declined from 1.173±0.156 to 0.540±0.076 (p<0.01), and kept the lower level until 12 hour (0.536±0.100, p<0.01). There were not obvious changes of mRNA level ofα1 andα3 isoform due to hypoxia in cortical slices.Hypoxic culture caused an increase in expressions ofα1 isoform protein in cultured cortical neurons, which is consent to its mRNA change. The levels of protein at 4 and 8 hour after hypoxia show a increasing trend which was not significant, but at 12 hour after hypoxia the level ofα1 isoform protein rose from 0.471±0.052 to 0.806±0.078 (p<0.01). The changes ofα3 isoform protein were similar to the decreases of its mRNA. The protein level at 4 hour after hypoxia decreased from 0.730±0.065 to 0.501±0.097 (p<0.05), and with prolonged hypoxic time it attenuated further from 0.446±0.077 at 8 hour after hypoxia to 0.448±0.109 at 12 hour after hypoxia (p<0.01), respectively. Changes in fluorescent intensity ofα1 andα3 isoform had the same trend as their protein expression. And there were not significant changes in protein levels ofα1 andα3 isoform in cortical slices at 15 min, 30 min and 60 min after hypoxia.Conclusion: Perfusing cortical slices with free-glucose and low oxygen and the hypoxic culture of cortical neurons simulate two different hypoxic injuries successfully. At the early stage of hypoxic exposure, the function of Na pump increases compensatoryly, but decreases step by step with prolonged hypoxic time. Hypoxic exposure downregulates the expressions of mRNA and protein of Na pumpα3 isoform and upregulates those ofα1 isoform. III Low-affinity Na pump involved in hypoxic injury in cortical neurons.Objective: This part of investigation was undertaken to understand the distinct functions of high- and low-affinity Na pump when suffering from hypoxia in cortical neuronsMethods: Hypoxic condition was simulated by perfusing free-glucose and low oxygen ACSF for different time.Changes of membrane current (?I/Cm) in neurons from cortical slices were determined at 0, 2, 4, 8 and 10 after hypoxia. We examined the membrane current through perfusion of both TTX (1μmol/L) and hypoxia or 6-min hypoxia before TTX in order to explore the relationship between ?I/Cm induced by hypoxia and activation of Na channel.We evaluated ?I/Cm by synchronously giving 10 min hypoxia and different concentrations of Oua (10 nmol/L, 100 nmol/L, and Oua 10μmol/L, which can excite high-affinity pump, inhibit high-affinity pump, and partially inhibit low-affinity pump respectively) and then calculated ?I/Cm at 0, 2, 4, 8, and 10 min after hypoxia, in order to understand the relationship between Na pump and hypoxia-mediated ?I/Cm.We examined fluorescent signals of [Ca2+]i and intracellular Na+ concentration ([Na+]i) in cultured cortical neurons using video based motion edge detection system (Fura-2 10μmol/L and SBFI 10μmol/L).①A change in ratio (?Ratio) of [Ca2+]i was determined by perfusing free-glucose and low oxygen HBS for 10 min, and calculated at 0, 2, 4, 6, 8 and 10 min after hypoxia.②?Ratio of [Na+]i and [Ca2+]i were determined by perfusing concentrations from 10-9 to 10-3 mol/L, and each concentration lasted for 2 min.③Cultured cortical neurons were pretreated by Oua of 100 nmol/L, 10μmol/L, or 1 mmol/L, and then suffered from hypoxia and Oua (the same concentration) synchronously.Results: Hypoxic exposure increased membrane current of neurons in cortical slices. Membrane current of neurons was enhanced time-dependently (r=0.98028,P<0.01) during the 10-min hypoxia exposure (P<0.01), and the increasing values were 0, -0.014±0.006, -0.028±0.013, -0.035±0.016, -0.044±0.022, -0.058±0.026 and -0.074±0.025 pA/pF, respectively, at 0, 2, 4, 6, 8 and 10 min after hypoxia, suggesting that the abnormal open of ionic channels was mediated by hypoxia.The increasing membrane current induced by hypoxia was completely blocked by TTX (1μmol/L) under condition without influences of Ca2+ and K+ channel. If cortical slices were pretreated by 6 min hypoxia and then suffered from both TTX and hypoxia, the increase of membrane current was not continued and kept the level at 6 min after hypoxia (-0.033±0.013 pA/pF), which was lower than that of alone hypoxia for 10 min (-0.058±0.028 pA/pF, P<0.01). These results indicate that increasing membrane current induced by hypoxia was TTX-sensitive Na current.Na pump affects the increase of membrane current induced by hypoxia. Oua 10μmol/L, partially inhibition of low-affinity pump, made membrane current increasing further, at 0, 2, 4, 6, 8 and 10 min after hypoxia the values of ?I/Cm were 0, -0.031±0.010, -0.041±0.013, -0.053±0.007, -0.057±0.008 and -0.069±0.012 pA/pF (P<0.05). By perfusing both Oua 100 nmol/L and 10 min oxygen-free A CSF, values of ?I/Cm displaying a decreasing extend were 0, -0.021±0.003, -0.028±0.006, -0.038±0.016, -0.047±0.018 and -0.055±0.020 pA/pF, respectively (P>0.05). However, values of ?I/Cm were obviously lower compared with each point of single hypoxia by perfusing both Oua 10 nmol/L and 10-min hypoxia, they were 0, -0.009±0.011, -0.011±0.015, -0.010±0.016, -0.009±0.018 and -0.012±0.020 pA/pF (P<0.01), which showed that 10 nmol/L Oua protected cortical neurons from hypoxic injury. Oua also affects the [Ca2+]i and [Na+]i in cultured cortical neurons.Inhibition of Na pump in cultured cortical neurons by Oua caused the increases in the [Ca2+]i and [Na+]i in a concentration-dependent manner in normal ACSF.ΔRatio of [Na+]i were 0.017±0.004, 0.030±0.004 and 0.035±0.004 (P<0.01) when perfusing Oua 10-5, 10-4 , and 10-3mol/L, which were higher compared with normal control.ΔRatio of [Ca2+]i also exhibited an significant elevate, which were 0.056±0.013, 0.107±0.018, 0.158±0.011, 0.185±0.016 and 0.192±0.014(P<0.05 or P<0.01) by perfusing Oua 10-7, 10-6, 10-5, 10-4, and 10-3 mol/L. These results suggest that inhibition of Na pump causes a raise in cortical neurons [Ca2+]i.Increase of [Ca2+]i mediated by hypoxia in cultured cortical neurons was time-dependent,ΔRatio of [Ca2+]i were 0, 0.0220±0.0012, 0.0693±0.0052, 0.1047±0.0148, 0.1397±0.0165 and 0.1637±0.0122, which were higher compared with control at 0, 2, 4, 6, 8 and 10 min point (P<0.01).Oua of 10 nmol/LdecreasedΔRatio of [Ca2+]i raised by hypoxia, and values ofΔRatio were 0, 0.0163±0.0009, 0.0300±0.0050, 0.0520±0.0141, 0.0710±0.0155 and 0.0983±0.0183, lower compared with control at 0, 2, 4, 6, 8 and 10 min point (P<0.05), suggesting that exciting high-affinity pump counterwork partially the increase of [Ca2+]i induced by hypoxia in cultured cortical neurons. Both hypoxia and Oua 10μmol/L perfusion did not change the increases in the [Ca2+]i induced by hypoxia, suggesting low-affinity pump involved mainly in hypoxic injury.When a higher level of the [Ca2+]i was caused by Oua of 1 mmol/L in cortical neurons, perfusion of hypoxic-ACSF contained Oua of 1 mmol/L could not make it further raising and kept the same level within 4-min hypoxia as them before hypoxia (P>0.05), this is, hypoxia-mediatedΔRatio of [Ca2+]i nearly equaled to 0, and much lower than these points of single hypoxia (P<0.01). However, with prolonged hypoxic time,ΔRatio of [Ca2+]i increased slightly, the values at 6, 8 and 10 min were 0.0200±0.0144, 0.0556±0.0141 and 0.0837±0.0128(P<0.05), which were lower than those in alone hypoxia.Conclusions: Hypoxia can cause Na+ entering cortical neurons and an increase of [Ca2+]i, the inhibition of Na pump also elevates the [Ca2+]i through increasing [Na+]i and the complete inhibition of Na pump can block hypoxia- mediated changes of [Ca2+]i in cortical neurons within 4 min hypoxic exposure, suggesting that Na pump involves in the effect of hypoxia on cortical neurons by regulating [Ca2+]i indirectly and that Na pump is one of targets at the early stage of hypoxic injury, but some other mechanisms involved in prolonged-time hypoxia. Exciting high-affinity pump protected cortical neurons from hypoxia injury.