| As an important neurotransmitter, excitatory amino acid has the effects of regulating the normal physiological functions of brain. Glutamate is the major excitatory neurotransmitter in the CNS. Excessive glutamate release may cause neuronal cell death in brain disorders by stimulating glutamate receptors (GluR), especially its ionotropic glutamate receptors (NMDA receptors).Glumate (Glu) released from neuron during brain ischemia is a main cause of inducing neuron death. When brain ischemia occurs, glutamate in brain will exceed its normal level due to energy failure and loss of ATP. The activity of Na+,K+-ATPase is decreased, and the water and Na+ will retain inside the membrane. As a result, overload of intracellular Ca2+ occur through the inverse mode of Na+-Ca2+ exchanger (NCX). The activations of Ca2+-related phosphatidase, protease, and endonuclease induce the decomposition of membrane phospholipid and the destruction of cystoskeleton, which are all the irreversible damages to the neuron.The Na+, K+-ATPase is a member of the P-type ATPase superfamily. It has multiple regulatory functions, including maintenance of cellular [Na+] and [K+] gradients, membrane excitability, as well as engaged in assembly of multiple protein complexes which transmit signals to different intracellular compartments. As a key enzyme in maintaining basic physiological function of cells, more and more researches are focused on the effect of Na+, K+-ATPase on the process of hypoxia. When Na+, K+-ATPase is inhibited during hypoxia, the Na+ will be retained inside the membrane, followed by an exchange of 2 Ca2+ from ectocellular and 3 Na+ from intracellular via NCX. This progress leads to calcium overload and cerebral edema.Our early researches have already proved, after hypoxia the excitatory amino acid will exceed its normal level, at the mean time the activity of Na+, K+-ATPase decreased apparently. Moreover, both mRNA level and protein content of theα2 and theα3 isoforms of Na+, K+-ATPase decreased, nothing changed aboutα1 isoform. These results indicated that the high affinityαisoform of the Na+, K+-ATPase involved in the brain ischemia.When brain ischemia happened, the function of the Na+, K+-ATPase was decreased, and glutamate would exceed its normal level and activate the NMDA receptors. Is there any relationship between them? In order to investigate the mechanisms of brain ischemic damages, we observed the influence of hypoxia on the excitatory amino acid current (NMDA current) in rat cortical neuron. Furthermore, we investigated whether Na+,K+-ATPase involved in the excess activation of NMDA receptors in rat cortical slices or not under hypoxia condition.Objective:Our main point is to see under normoxia and hypoxia conditions whether the Na+,K+-ATPase involves in the change of the excitatory amino acid in rat cortical neuron and its mechanism.Method: We used SD rats, 12-16 days old, to prepare for brain slices in usual ways. The whole-cell mode was used to examine NMDA current by the patch clamp systems with infrared differential interference contrast (IR-DIC) optics. Our experiment is focused on the effects of hypoxia, AP-5 or MK-801, and different concentrations of dihydroouabain (DHO) on NMDA current.Results:1 Assessment of NMDA current in rat cortical neuronWe recorded an inward movement of holding current, defined as the NMDA current, which was inhibited by perfused with AP-5 or MK-801, and the inhibitory rates of AP-5 MK-801 were 94.95% and 89.17%, respectively. These results suggested that the inward current we recorded was surely NMDA curent.2 The stability of NMDA current in rat cortical neuronThe NMDA currents recorded repeatedly in cortical neuron within 20 minutes were almost the same. This indicated that rat cortical neuron have a stable reaction to NMDA. So our experiments were finished in 20 minutes.3 Effect of hypoxia on the NMDA current in cortical neuron Under normoxia condition, NMDA 10μmol/L, 50μmol/L and 80μmol/L could cause an inward NMDA current in cortical neuron and the NMDA current densities were 3.557±0.393pA/pF, 6.987±0.509pA/pF and 7.226±0.558 pA/pF, respectively. After hypoxia, the NMDA current densities were 4.026±0.546pA/pF, 8.558±0.775pA/pF and 10.066±1.242pA/pF respectively. These results indicated hypoxia could cause an increase in NMDA current, and the increased level relied on the concentrations of NMDA.We also found that after perfused neuron with anoxia fluids for 5 minutes, the holding current had an inward movement, which could also be blocked by AP-5 or MK-801. AP-5 (100μmol/L) could diminish the current density from 0.272±0.144pA/pF to 0.019±0.007pA/pF, the inhibitory rate was 91.26%. MK-801 (100μmol/L) could diminish the current density from 0.252±0.097pA/pF to 0.025±0.012pA/pF, the inhibitory rate was 90.13%. Moreover, when we used 1μmol/L NMDA to pre-perfused the cortical neuron, hypoxia could cause a larger inward movement and the current density increased from 0.211±0.079pA/pF to 0.555±0.195pA/pF. This could also be blocked by AP-5 or MK-801. AP-5 (100μmol/L) diminished the current density from 0.543±0.32pA/pF to 0.031±0.039pA/pF, the inhibitory rate was 92.10%. MK-801 (100μmol/L) diminished the current density from 0.62±0.079 pA/pF to 0.056±0.009 pA/pF, the inhibitory rate was 90.84%. All of these results indicated hypoxia could bring an NMDA current, which might be caused by an extra release of endogenous excitatory amino acid, and the change of NMDA current induced by hypoxia was related to the level of excitatory amino acid before hypoxia.4 Effect of DHO on the NMDA current in cortical neuronUnder either normoxia or hypoxia conditions, after perfusion with DHO, the NMDA current was decreased. Besides, the NMDA currrent became smaller and the inhibitory rate went higher along with the increase of DHO concentrations (from 10-11 mol/L to 10-3mol/L). Under normoxia condition, after perfused with different concentrations of DHO (from 10-11 mol/L to 10-3mol/L) the NMDA curent densities were: 7.081±0.798pA/pF, 7.064±0.909pA/pF, 6.290±0.677 pA/pF, 5.097±0.909pA/pF, 4.18±1.104pA/pF, 3.606±1.027pA/pF, 3.045±0.583pA/pF, 2.003±0.68pA/pF and 1.672±0.548pA/pF. The inhibitory rates were: 2.06%, 2.30%, 13.00%, 29.50%, 42.18%, 50.13%, 57.89%, 72.29% and 76.88%. Under hypoxia condition, after perfused with DHO (from 10-11 mol/L to 10-3mol/L) the NMDA curent densities were: 10.033±0.459pA/pF, 9.772±0.531 pA/pF, 8.808±1.019pA/pF, 7.254±0.998pA/pF, 6.401±0.649pA/pF, 6.054±0.724pA/pF, 5.737±0.616pA/pF, 5.169±0.666pA/pF and 4.978±0.495pA/pF. The inhibitory rates were: 0.32%, 2.91%, 12.50%, 27.94%, 36.41%, 39.85%, 43.00%, 48.64% and 50.55%. These datas demonstrated Na+, K+-ATPase involved in the regulation of NMDA current, and the function of the Na+, K+-ATPase was related to the NMDA current closely. However, after hypoxia, NMDA current density was much larger than normoxia after perfused with different concentrations of DHO, which indicated the regulation of NMDA current of cortical neuron after hypoxia may depend mainly on the release of excitatory amino acid, not the function of the Na+, K+-ATPase.5 Effect of vanadate on the NMDA current in cortical neuron1μmol/L and 1mmol/L vanadate could block the NMDA current significantly just like DHO. The inhibitory rates were 49.25% and 76.4%. This demonstrated the regulation of NMDA current was related to the function of the Na+,K+-ATPase.6 Mechanism of the NMDA current regulation by sodium pumpThe fit curve of the NMDA current regulated by DHO demonstrated there were two different functional Na+, K+-ATPase on rat cortical neuron, the high and the low affinity pump. The fit parameters under normoxia condition were: kh=3. 8143×10-9, kl=1.3671×10-5, fh=0.5839, fl=0.4161. Under hypoxia condition: kh=2.3311×10-9, kl=1.1735×10-5, fh=0.7353, fl=0.2647. By analyzing the fit curve we found: both the high and the low affinity pump involved in the regulation of NMDA currrent. But the hypoxia affects mainly the high affinity pump, not the low affinity pump.NCX inhibitor did not affect Cortical neuron's NMDA current density, which were 7.649±1.224pA/pF and 7.102±1.226pA/pF before and after perfused with KB-R7943 (10μmol/L) (P>0.05). However, NMDA current density decreased from 8.309±2.513pA/pF to 2.173±1.412pA/pF (P<0.01) after perfused with genistein (100μmol/L), the inhibitory rate was 74.81%. The mechanism of the regulation of NMDA current was closely correlated with signal transduction function of the Na+, K+-ATPase.Conclusion:1 Hypoxia could either directly induce NMDA current or could significantly increase NMDA current induced by NMDA in rat cortical neuron, and the induced or increased NMDA current densities are markedly the concentration-dependent to NMDA.2 Under either normoxia or hypoxia conditions, DHO inhibits the NMDA current of the cortical neurons in a concentration-dependent manner, implying Na+,K+-ATPase involves in the regulation of NMDA current in the cortical neurons, and the level of the Na+,K+-ATPase activity is closely related to the degree of the NMDA current.3 Hypoxia can diminish the inhibitory effect of DHO on the NMDA current, and mainly changes the correlative parameter of the high affinityαsubunit of the Na+,K+-ATPase. This indicates the regulation of hypoxia on NMDA current maily depends on the high affinity pump.4 NCX inhibitor KB-R7943 does not influence the NMDA current density, but PTK inhibitor genistein significantly decreases the NMDA current density. These results indicate that the signal transduction function, not the ionic pump function of Na+,K+-ATPase has a key role in regulation of the NMDA current in the cortical neurons.
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