| 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, skeletal muscles, 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 pyramidal cells from cortical slices of rat.Objectives: This part of investigation was undertaken to characterize the Na pump current in neurons from cortical slices.Methods: The young SD rats (13–16 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 -65 mV, at first we identified that the recording cell was a neurons by membrane depolarization (-65mV—0mV—-65mV) to evoke Na+ channel current, and then the bath solution was switched from normal ACSF to the perfusing solution contained Ouabain (Oua, 10-1010-3 mol/L), which resulted in a inward shift of the membrane current at the whole cell mode. Then Oua-sensitive Na pump currents (IP) were calculated to changes rate (ΔIP) according to the amplitude of Na pump current evoked by 10-3 mol/L Oua, and theΔIP-[Oua] curve was drew with the rate as ordinate and concentration of Oua ([Oua]) as abscissa. The whole experiment was executed under the conditions of room temperature.Results: Oua-sensitive current represented Na pump current because the amplitude evoked by 1 mmol/L Oua equaled to that induced by free-K+ perfusing solution.Na pump currents were recorded in 53 neurons in the study, and IP-[Oua] curve could be disported into 3 patterns based on the neuron's response to 10-8 mol/L Oua. The 3 patterns included exciting, inhibitory and ineffective patterns and the 3 parts of cells in the experiments were pyramidal neurons because there were not obvious difference in Na+ current, cell diameter and capacitance (Cm).In theΔIP-[Oua] relation curve of exciting pattern (in 15 from 53 neurons, 28.3%), theΔIp values produced by each concentration of Oua from 10-10 to 10-3 mol/L were -0.014±0.042, -0.049±0.071 -0.067±0.099, -0.111±0.063, -0.141±0.089, -0.300±0.067, -0.437±0.094, -0.690±0.106 and -1.000±0.000, respectively. The curve was well fitted using a three-binding site model and the dissociation constants for high-affinity exciting binding site, high-affinity inhibitory binding site, and low-affinity inhibitory binding site were 1.391 nmol/L, 8.506 nmol/L, and 216.1μmol/L.In theΔIP-[Oua] relation curve of inhibitory pattern (in 33 from 53 neurons, 62.3%), all theΔIp values on the curve (Oua concentrations from 10-10 to 10-3 mol/L) were 0.016±0.063, 0.042±0.089, 0.065±0.107, -0.071±0.100, -0.135±0.042, -0.272±0.059, -0.467±0.078, -0.638±0.038 and -1.000±0.000, respectively. 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 60.5 nmol/L and 202.3μmol/L. In theΔIP-[Oua] relation curve of ineffective pattern (in 5 from 53 neurons, 9.4%), Oua from 10-10 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 198.2μmol/L.The high-affinity pumps generated 19.620.0% of the total Na pump current and very likely included theα2 andα3 isoform. The low-affinity pump corresponding to 80.080.4% of the total Na pump current attributed toα1 isoform.Conclusion: Cortical pyramidal neurons express two functionally distinct pump as expected for high-affinity pump and low-affinity pump. II Na pump involves in hypoxic injury in cortical pyramidal cells of ratObjective: 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: Neurons were exposed to oxygen glucose deprivation by incubating cortical slices or cultured cortical neurons with free-glucose and low-oxygen solution. In order to determine whether the two hypoxic methods were successful, TTC staining was employed to evaluate activity of cortical slices after hypoxia. And we examined changes of membrane current density (?I/Cm) in pyramidal cells of cortical slices and changes of intracellular Ca2+ concentration (?Ratio of [Ca2+]i) after hypoxia to estimate the degree of hypoxic injury of neurons.Effects of Na pump on ?I/Cm induced by hypoxia in cortical slices. We evaluated ?I/Cm by synchronously giving 10 min hypoxia and different concentrations of Oua (10 nmol/L and Oua 10μmol/L, which can excite high-affinity pump or inhibit high-affinity pump, and inhibit low-affinity pump respectively) and then calculated ?I/Cm at 0, 2, 4, 8, and 10 min after hypoxia.Effects of Na pump on ?Ratio of [Ca2+]i induced by hypoxia in cultured cortical neurons. We examined fluorescent signals of [Ca2+]i and [Na+]i in cultured cortical neurons using video based motion edge detection system (Fura-2 10μmol/L and SBFI 10μmol/L).①?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.②Cultured cortical neurons were pretreated by Oua of 10 nmol/L or 10μmol/L, and then suffered from hypoxia and Oua (the same concentration) synchronously, and then calculated ?Ratio of [Ca2+]i at 0, 2, 4, 8, and 10 min after hypoxia.Effects of hypoxia on total Na pump current. Cortical slice were suffered hypoxia for 400s or 600s and then the total IP was measured.Results: TTC staining showed that the activity of cortical slices reduced significantly (P<0.01) to its 72.6% primary activity after hypoxia for 5 min, to 67.3% after hypoxia for 10 min and to 65.2% after hypoxia for 15 min, but there was not obvious difference among 5-min, 10-min and 15-min hypoxia (P>0.05). Membrane current of pyramidal cells of cortical slices changed little for 10 min at normoxia, but ?I/Cm increased significantly and time-dependently (r=0.98028, P<0.01) after 10-min hypoxic exposure (P<0.01), and the increasing values were 0, -0.015±0.006, -0.026±0.011, -0.031±0.016, -0.041±0.010 and -0.055±0.014 pA/pF, respectively, at 0, 2, 4, 6, 8 and 10 min after hypoxia, the difference was significant compared with normoxia (0-min hypoxia) after hypoxia lasting for 4 min (P<0.01). Increase of [Ca2+]i mediated by hypoxia in cultured cortical neurons was time-dependent (r=0.973, p<0.01),ΔRatio of [Ca2+]i were 0, 0.019±0.001, 0.048±0.006, 0.089±0.008, 0.130±0.015 and 0.165±0.008, which were higher compared with control at 4, 6, 8 and 10 min point (P<0.05 or 0.01).△I/Cm enhanced obviously (P<0.01) induced by Oua 10μmol/L, and then increased further. The degree of increase was time-dependently (r=0.908, P<0.01) and significantly. ?I/Cm at 2, 4, 6, 8 and 10 min after hypoxia were higher compared with normoxia (P<0.05 or 0.01) and each point of single hypoxia (P<0.05 or 0.01).On pyramidal cells that had exciting-binding site, ?I/Cm of cells of exciting part at 2, 4, 6, 8 and 10 min after hypoxia was unchanged compared with normoxia (P>0.05), but values at 4, 6, 8 and 10 min after hypoxia were dropped significantly compared with ach point of single hypoxia (P<0.05 or 0.01), which suggested that exciting high-affinity Na pump protected neurons from hypoxic injury. However on pyramidal cells that had inhibitory-binding site, ?I/Cm of cells of inhibitory part after 10-min hypoxia rose time-dependently (r=0.956, P<0.01) and significantly (P<0.01). The values at 4, 6, 8 and 10 min after hypoxia were unchanged compared with each point of single hypoxia (P>0.05), which suggested that inhibiting low-affinity Na pump had no effect on hypoxia injury. When the data of all cells in 2 parts were calculated together, the results showed that Oua 10 nmol/L could protect neuron due to increasing degree of ?I/Cm after 8-min hypoxia was obviuosly lower than single hypoxia (P<0.05 or 0.01).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 normoxic solution (r=0.984, P<0.01).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 0.01) by perfusing Oua 10-710-3 mol/L. These results suggest that inhibition of Na pump causes a raise in cortical neurons [Ca2+]i. Pretreated with 10 nmol/L Oua, ?Ratio of [Ca2+]i in cultured neurons was raised by 4-min, 8-min and 10-min hypoxia significantly (P<0.05 or 0.01), but increasing degree of ?Ratio after 10-min hypoxia was obviously lower than single hypoxia (P<0.05). And Pretreated with Oua 10μmol/L, increasing degree of ?Ratio after 10-min hypoxia was obviously higher than single hypoxia (P<0.05 or 0.01).After cortical slices suffered from hypoxia for 400s or 600s, Ip/Cm decreased significantly (P<0.01), but there was no difference between 400s and 600s hypoxia (P>0.05).Conclusions: Hypoxia can cause an increase of [Ca2+]i, the inhibition of Na pump also elevates the [Ca2+]i through increasing [Na+]i. Exciting high-affinity Na pump can protect neurons from hypoxic injury and inhibiting low-affinity Na pump can aggravate the degree of hypoxic injury. III Effects of ATP and H2O2 on Na pump in hypoxic or normoxic pyramidal cells from rat cortex.Objective: This part of investigation was undertaken to understand how ATP and H2O2 affect differently on Na pump in hypoxic injury of pyramidal cells of rat cortex.Methods: Effects of ATP on Na pump All the cortical slices were divided into 2 parts, which were treated with adequate ATP or without any ATP in pipette solution respectively. Every part was divided into 2 groups of control and hypoxia. In control group, the bath solution was switched from normoxic solution to the solution contained Ouabain (Oua, 10-1010-3 mol/L), and then calculated Ip. In hypoxia group, cortical slices were exposed to hypoxia and subsequently perfused with low-oxygen solution contained Ouabain (Oua, 10-1010-3 mol/L), and then calculated Ip'. ThenΔIP-[Oua] curve was drawn according to the amplitude of Na pump current evoked by 10-3 mol/L Oua. Therefore there were 4 groups which were control-ATP, hypoxia-ATP, control-no ATP and hypoxia-no ATP, respectively.Effects of H2O2 on Na pump Pretreated with 100 or 300μmol/L H2O2 for 200s, the bath solution was switched to the solution contained H2O2 and Ouabain (Oua, 10-1010-3 mol/L), and then calculated Ip. Then Oua-sensitive curve was draw according to the amplitude of Na pump current evoked by 10-3 mol/L Oua.Translocations of Na pumpαsubunit Cortical slices were exposed to hypoxia, 100 or 300μmol/L H2O2 for 10 min, and then the total or membrane proteins were extracted. We examined changes of protein of Na pumpαsubunits through Western Blot.Results: Effects of ATP on Na pump When there was not any ATP in pipette solution, Na pump current density (IP/Cm) decreased significantly (P<0.05) induced by 400s or 600s hypoxia. IP/Cm dropped from 0.365±0.072 pA/pF of normoxia to 0.282±0.025 and 0.272±0.044 pA/pF (P<0.01) respectively. IP/Cm of hypoxia-no ATP group induced by Oua decreased significantly compared with control-no ATP group (p<0.05 or p<0.01), which showed that functions of high- and low-affinity Na pump were inhibited due to lack of ATP in neurons.ΔIP-[Oua] curves was fitted for control-no ATP group and hypoxia-no ATP group and the dissociation constants for high-affinity Na pump in hypoxia-no ATP group was 0.1471 nmol/L, which was obviously lower than that in control-no ATP group (0.1732 nmol/L) and was 84.93% of control-no ATP group. However the dissociation constants for low-affinity Na pump in hypoxia-no ATP group was 170.9μmol/L, which was higher than that in control-no ATP group (134.7 nmol/L) and was 1.52 times of control-no ATP group. Moreover, the proportion of high-affinity Na pump of hypoxia was 20.53%, which was 1.52 times of control-ATP group (13.47%), and the proportion of low-affinity Na pump of hypoxia was 79.47%, which was 0.9184 times of control-ATP group (86.53%). Results showed that hypoxia enhanced composing proportion and sensitivity to Oua of high-affinity Na pump.However, when there was adequate ATP in pipette solution, IP/Cm had no change induced by 400s or 600s hypoxia. IP/Cm of hypoxia-ATP group induced by 10-10～10-3 mol/L Oua had unchanged compared with control-ATP group (p>0.05).ΔIP-[Oua] curves was fitted for control-ATP group and hypoxia-ATP group and the dissociation constants for high- and low-affinity Na pump of hypoxia-ATP group were 0.2888 nmol/L and 91.94μmol/L, which were obviously lower than those in control-ATP group (95.38 nmol/L and 147.6μmol/L) and were 0.303% and 62.29% of control-ATP group. The results showed that the sensitivity of high- and low-affinity Na pump to Oua increased, especially for high-affinity Na pump. Moreover, the proportion of high-affinity Na pump of hypoxia was 15.88%, which was 1.211 times of control-ATP group (12.49%), and the proportion of low-affinity Na pump of hypoxia was 84.12%, which was 0.9601 times of control-ATP group (87.51%). Results shows that hypoxia increased composing proportion of high-affinity Na pump, and raised sensitivity of high- and low-affinity Na pump to Oua, especially high-affinity Na pump.IP/Cm in hypoxia-no ATP group induced by 10-8, 10-5, 10-4 and 10-3 mol/L Oua decreased significantly compared with that in control-ATP group (p<0.05 or 0.01), IP/Cm in control-no ATP group induced by 10-5, 10-4and 10-3 mol/L Oua decreased significantly compared with that in hypoxia-ATP group (p< 0.01), and IP/Cm in hypoxia-no ATP group induced by 10-8, 10-6, 10-5, 10-4 and 10-3 mol/L Oua decreased significantly compared with hypoxia-ATP group (p< 0.01), which showed that lack of ATP in neurons inhibited low-affinity Na pump function. According to fitting lines, the affinity to Oua of high-affinity Na pump among 4 groups changed significantly. The dissociation constant for high-affinity Na pump of hypoxia-no ATP group was lowest and 648 times of control-ATP group, which showed that the sensitivity to Oua was maximal. But the dissociation constant for low-affinity Na pump of hypoxia-no ATP group was most which showed that the sensitivity to Oua was lowest.Effects of H2O2 on Na pump As perfusing H2O2 100μmol/L and Oua simultaneously, IP/Cm induced by 10-1010-3 mol/L Oua changed inapparently compared with that in control (p>0.05), but as perfusing H2O2 300μmol/L and Oua simultaneously, IP/Cm induced by 10-6, 10-5 and 10-3 mol/L Oua reduced significantly compared with that in control (p<0.05 or 0.01). IP/Cm of high- and low-affinity Na pump was dissociated from the total IP/Cm respectively, results showed that H2O2 300μmol/L inhibited high-affinity Na pump current mainly (p<0.01), and had no effect on low-affinity Na pump current.ΔIP-[Oua] curve was fitted and the dissociation constant for high-affinity Na pump under actions of H2O2 100μmol/L was 0.2125 nmol/L, which was obviously lower than control (95.38 nmol/L) and was 0.22% of control, and it under actions of H2O2 300μmol/L of 0.0582 nmol/L and was 0.6102‰of control, which showed that the sensitivity of high-affinity Na pump to Oua was increased obviously by H2O2. The effect of H2O2 on low-affinity Na pump was less than on high-affinity Na pump. The dissociation constant under actions of H2O2 100μmol/L was 87.32μmol/L and was 59.2% of control, and it actions of H2O2 300μmol/L was 58.21μmol/L and was 39.4% of control, which showed that the sensitivity of low-affinity Na pump to Oua was increased obviously by H2O2. Moreover, the proportions of high- and low-affinity Na pump under actions of H2O2 were changed, they were 12.4% and 87.6% for control, under actions of H2O2 100μmol/L they were 10.8% and 89.2%, and under actions of H2O2 300μmol/L they were 3.3% and 96.7%.Translocations of Na pumpαsubunit Total protein levels of 3 types ofαsubunit of Na pump and membrane protein levels ofα1 andα3 subunit kept the primary quantity after 10-min hypoxic incubation (p>0.05), but the membrane protein level ofα3 subunit after hypoxia increased significantly to 0.672±0.041 compared with control (0.472±0.060) (p<0.01). Total protein and membrane protein levels kept the primary quantity after 10-min incubation with H2O2 100 or 300μmol/L (p>0.05).Conclusions: Lack of ATP in neurons inhibits low-affinity Na pump function, and has no effect on high-affinity Na pump. Reactive oxygen species inhibit high-affinity Na pump function, and have no effect on low-affinity Na pump. Both lack of ATP and reactive oxygen species can raise the sensitivity of high-affinity Na pump to Oua obviously. The proportion of high-affinity Na pump to total Na pump enhances significantly, andα3 subunit translocates from cytoplasm to membrane. |
