胞内Ca2+、Na+和ATP的稳定性介导NMDA诱导兴奋保护作用
作者：林穗珍 Robert P. Irwin 李晓瑜 颜光美
单位：林穗珍 李晓瑜 中山医科大学附属第一医院儿科；颜光美 药理教研室； 广州， 510080；林穗珍 Robert P. Irwin 颜光美 美国印第安纳大学医学院药理、毒理系； 印第安纳波利斯， 46202)
关键词：谷氨酸；神经元；钙；钠；MK801；兴奋性氨基酸类；N-甲基-天冬氨酸；神经保护药
中山医科大学学报990403Intracellular Ca2+, Na+, and ATP Homeostasis Mediated NMDA-induced Excitoprotection in Rat Cerebellar Granule Neurons
Lin Suizhen1,3 Robert P. Irwin3 Li Xiaoyu1 Yan Guangmei2,3
, 百拇医药
( 1 Department of Pediatrics, First Affiliated Hospital 2 Department of Pharmacology, Sun Yat-sen University of Medical Sciences,Guangzhou, 510089; 3 Department of Pharmacology and Toxicology, Indiana University,School of Medicine, Indianapolis, IN 46202)
Abstract Objective:To study the mechanisms underlying N-methyl-D-aspartate (NMDA)-induced excitoprotection in cultured rat cerebellar granule neurons. Methods: Intracellular Ca2+ ([Ca2+]i) and intracellular Na+([Na+]i) were measured microspectrofluorimetrically with the Ca2+ sensitive dye fura-2/Am and the Na+-sensitive indicator SBFI/AM respectively. Cytosolic denosine triphosphate(ATP) was quantified by means of reverse-phase HPLC. Results: First. Glutamate rapidly induced a peak [Ca2+]i response in both untreated and NMDA treated neurons. In NMDA treated neurons, [Ca2+]i then rapidly dropped to a stable plateau concentration above that of the basal condition. Thirty minutes later, with the removal of glutamate, [Ca2+]i rapidly returned to near basal concentrations. In contrast, with NMDA-untreated neurons, prolonged glutamate exposure resulted in a heterogeneous [Ca2+]i response in many neurons,with [Ca2+]i either gradually or abruptly increasing. Upon glutamate removal, many untreated neurons failed to return to basal levels. Second,Glutamate rapidly induced a peak [Na+]i response in both untreated and NMDA-treated neurons. However, the glutamate-induced [Na+]i peaks, during and after glutamate were all higher in untreated neurons, compared with NMDA-pretreated neurons. Third, Glutamate induced the consumption of ATP stores in the neurons. However,ATP consumption in the neurons pretreated with NMDA was significantly reduced,compared with neurons not pretrect. Conclusions:NMDA-induced excitoprotection in cerebellar granule neurons is associated with enhanced [Ca2+]i and [Na+]i homeostasis by reducing ATP consumption.
, 百拇医药
Subject headings glutamate; neurons; calcium; sodium; MK801; excitatory amino acids, N-methylaspartate; neuroprotective agents
摘 要 目的：研究N-甲基-D-天冬氨酸(NMDA)诱导大鼠小脑颗粒神经元兴奋毒性保护作用的机制。方法：分别用Ca2+和Na+敏感的染料Fura-2/AM和SBFI/AM测定胞内Ca2+和Na+浓度[(Ca2+)i,(Na+)i]。并用反向高效液相色谱法测定胞内三磷酸腺苷(ATP)的含量。结果：①在NMDA预处理组与非处理组，谷氨酸均迅速触发胞内Ca2+反应高峰，两者无显著性差异。而后在NMDA处理组，胞内Ca2+迅速下降，并维持在高于静息Ca2+水平的平台状，撤离谷氨酸后，[Ca2+]i迅速下降并恢复至静息水平；而NMDA非处理组则相反。②谷氨酸均诱发上述两组神经元[Na+]i升高，在NMDA非处理组，[Na+]i均高于NMDA预处理组。③谷氨酸消耗胞内ATP， 而NMDA预处理组则减少胞内ATP的耗竭。结论：NMDA诱导兴奋毒性保护作用是通过减少ATP的消耗而增强胞内Ca2+和Na+的自稳态。
, 百拇医药
中图号 R 979
Glutamate, the major excitatory neurotransmitter, is involved in many important CNS functions. However, it can also be an excitotoxin and is thought to be involved in the pathogenesis of neuronal death due to cerebral ischemia and trauma. Application of toxic concentrations of glutamate to cerebellar granule neurons can produce excessive increases in intracellular calcium ([Ca2+]i) and results in cell death[1]. Over stimulation of both N-methyl-D-aspartate (NMDA) and non-NMDA receptor subtypes may be involved in the excitotoxicity of glutamate. Exposure of cultured rat cerebellar granule neurons to subtoxic NMDA concentration has previously been shown to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations demonstrated by reduced susceptibility to glutamate[2]. Also,we showed that subtoxic concentrations of NMDA could shift the concentration-response for glutamate toxicity in a concentration and time-dependent manner. This effect has been shown to be dependent on protein synthesis[3]. In this study we have further characterized intracellular excitoprotection in cerebellar granule neurons including: changes in intracellular calcium([Ca2+]i), intracellular sodium ([Na+]i) homeostasis and alteration of the energy metabolic state.
, 百拇医药
1 Materials and Methods
1.1 Materials
L-Glutamate, NMDA, and MK-801 were from RBI. Fura-2/AM and SBFI/AM were from molecular probes. Ionomycin was from calbiochem. Ethylene imine polymer was from fluka chemical corp. All other chemicals were purchased from Sigma.
1.2 Primary neurons culture
Rat cerebellar granule neurons were prepared from 8-day-old Sprague-Dawley rat pups as described previously[3]. Briefly, neurons were dissociated from freshly dissected cerebellar by mechanical disruption in the presence of trypsin and DNase, and then plated in poly-1-lysine-coated dishes. Cells were seeded at a density of (1.5～1.8)×106 cells/mL in basal modified Eagle′s medium (BME) containig 10% fetal bovine serum and 25 mmol/L KCl. Cytosine arabinoside (10 μmol/L) was added to the culture medium after 24h to arrest the growth of non-neuronal cells. D-Glucose (5 mmol/L) was added to the cultures on day 7 and every fourth day thereafter.
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1.3 Measurement of Intracellular Ca2+
Measurement of [Ca2+]i was carried out as previously described using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2[4]. In brief, NMDA (50 μmol/L) was added to the culture medium of pretreated neurons for 24 h. Neurons were incubation with 5 μmol/L Fura-2 for 45 min. Cells were then washed twice with BME containing 25 mmol/L KCl and 10 μmol/L glycine. The cells were continuously perfused with same buffer at 37 ℃. Following that [Ca2+]i was measured for 5 min as a basal [Ca2+]i of neurons, glutamate then was perfused for 30 min. The same buffer was then used to perfuse for another 30 min to wash out glutamate. Neurons were illuminated on an inverted microscope (Nikon Diaphot). Excitation of fura-2 was at 340 and 380 nm. Cell images were digitized and stored for subsequent analysis (Universal Imaging Co, USA). Calculation of [Ca2+]i was carried out as described by Grynkiewicz et al[5] .
, 百拇医药
1.4 Measurement of Intracellular Na+
Measurement of [Na+]i was carried out as previously described using microspectrofluorimetry and the Na+ sensitive indicator SBFI/AM[6]. Briefly, cells was loaded with SBFI/AM(10 μmol/L) and returned to the incubator for 45 min. Cells were washed 3 times and allowed ≥15 min to complete the hydrolysis of the ester linkage before starting imaging. Cells were imagined using identical conditions to that for fura-2 experiments with excitation wavelengths, 340 nm and 380 nm and emitted light monitored at 510 nm. Background intensity levels were subtracted and in situ calibrations were carried out as descried by Harootunian et al[7] .
, 百拇医药
1.5 Measurement of ATP
Untreated and NMDA-treated neurons were exposed to glutamate (100 μmol/L) for 30 min. Cytosolic contents were then extracted with HCl and adenosine phosphonucleotide ([ATP]) levels were quantified with RP-HPLC before exposure to glutamate, following glutamate and removal glutamate[8].
1.6 Statistical Analysis
Data are presented as
±s. Unless otherwise stated, statistical comparisons were made using studentt-test.
, 百拇医药
2 Results
2.1 NMDA-induced excitoprotection enhances [Ca2+]i homeostasis
Since glutamate-induced cell death is related to increase in [Ca2+]i[1], we measured changes in [Ca2+]i associated with a 30 min exposure to glutamate in NMDA-pretreated and untreated cerebellar granule neurons in cultures. In NMDA-pretreatment of granule neurons resulted in a lessees reduction in basal [Ca2+]i but glutamate rapidly induced a peak [Ca2+]i response in both untreated and NMDA treated neurons. In NMDA treated neurons, [Ca2+]i then rapidly lowered to a stable plateau concentration above that of the basal condition. Thirty minutes later with the removal of glutamate,[Ca2+]i rapidly returned to near basal concentrations. In contrast, in case of untreated neurons prolonged glutamate exposure resulted in a heterogeneous [Ca2+]i response with many neurons gradually or abruptly increased [Ca2+]i. Upon removal of glutamate many untreated neurons failed to return to basal levels (Fig 1).
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Fig.1 NMDA-induced excitoprotection enhances [Ca2+]i homeostasis
[Ca2+]i was measured with the fluorescent Ca2+ indicator Fura-2. A, following recording basal [Ca2+]i, glutamate 100 μmol/L was applied for 30 min to cerebellar granule neurons pretreated for 24 h with NMDA (50 μmol/L). The experiment was terminated 30 min following application of glutamate. Each line (-) represent the calculted [Ca2+]i concentration of one cerebellar granule neuron.B, as in(A) but cerebellar granule neurons were untreated with NMDA (n =30 in each condition)
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To assess the role of the glutamate receptor subtypes in the NMDA-induced changes in Ca2+ homeostasis, we used the noncompetitive NMDA antagonist MK-801 to block the NMDA component of the glutamate-induced rise in [Ca2+]i. Experiments were performed as those with glutamate exposure alone except MK-801 (1 μmol/L) was perfused over the neurons for 2 min before addition of glutamate. MK-801 markedly reduced the glutamate-induced rise in [Ca2+]i and eliminated the differences responce between untreated and NMDA pretreated neurons in the 30 min during and after glutamate exposure.
, 百拇医药
2.2 NMDA-induced excitoprotection enhances [Na+]i homeostasis
To examine the effects of NMDA pretreatment on intracellular Na+ ([Na+]i) homeostasis, we measured [Na+]i using the Na+ sensitive indicator SBFI/AM.[Na+]i of cerebellar granule neurons was monitored before, during and following exposure to 100 μmol/L glutamate. Glutamate-induced a rapid elevation of [Na+]i from basal levels(Fig 2).
, 百拇医药
Fig. 2 NMDA-pretreatment of cerebellar granule neurons attenuates glutamateinduced eleva-
tion of intracellular Na+
c([Na+]i) was measured with the fluorescent Na+ indicator SBFI/AM. The drug application as described in Fig1. Straight-line (-) represent the mean of calculated [Na+]i concentration of NMDA-untreated cerebellar granule neurons. Circle line (-o-) represent the mean of calculated [Na+]i concentration of preexposed to NMDA (50 μmol/L) for 24 h (n =161 in each contion)
, 百拇医药
In both untreated and NMDA pretreated neurons, glutamate-induced a stable plateau concentration of [Na+]i above that of the basal condition. However, the magnitude of the response differed greatly between these groups. The glutamate-induced [Na+]i peak, during glutamate and the post glutamate (30 min) were both greater in the untreated than in the NMDA pretreated neurons.
2.3 NMDA-pretreatment effects on glutamate-induced changes of ATP content
, 百拇医药
ATP content was determined on cerebellar granule neurons before glutamate, following a 30 min exposure to glutamate (100 μmol/L) and 30 min following the washout of glutamate. NMDA treatment did not alter the mean ATP (% of control) content. Exposure to glutamate induced a consumption of ATP store. However, pretreatment with NMDA reduced the consumption of ATP (18% difference) than observed in untreated cells. Even a larger degradation (28% difference) follows a 30 min recovery period in untreated cells (Fig 3).
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Fig.3 NMDA-pretreatment of granule neurons reducedglutamate-induced depletion of ATP content
Data are from a representative experiment repeated three times with qualitatively similar results. Each point represents a mean of four replicates.*Statistically significant difference (P<0.05) between. NMDA-pretreated and untreated
3 Discussion
In the present study we have extended our previous ly findings that preincubation of cultured cerebellar granule neurons with subtoxic concentrations of NMDA for 24 h results in the induction of exitoprotective state as measured by subsequent exposure to glutamate. We found that NMDA pretreatment-induced alterations of Ca2+ and Na+ homeostasis in cerebellar granule neurons. Since MK-801 can completely block glutamate toxicity at the concentrations used in this study[3], could the NMDA-induced alterations of Ca2+ homeostasis in cerebellar granule neurons be simply due to “down-regulation” of NMDA receptors? We previous reported that exposure of NMDA results in no change of specific [3H] MK-801 binding to NMDA receptors and the immediate peak response in [Ca2+]i induced by glutamate was unaltered in cerebellar granule neurons preexposed to NMDA. Additionally, NMDA receptor-mediated neuroprotection extends to MPP+ toxicity in cerebellar granule neurons[9]. It is suggested that the NMDA-induced excitoprotection and maintain of Ca2+ homeostasis in cerebellar granule neurons is not due to “down-regulation” of NMDA receptors. Under both basal and pathological conditions, intracellular free Ca2+ concentration is stringently controlled because excessive Ca2+ influx is lethal to neurons. The transmembrane Na+ gradient is also tightly regulated; excessive Na+ influx which occurs under excitotoxic or ischemic conditions, may lead to inordinate excitatory amino acid efflux, as well as osmotic imbalance, cell swelling, and lysis[10]. Our data show that glutamate-induced influx of Na+ into the cerebellar granule neurons both in NMDA treated and untreated neurons, similar pattern was observed with Ca2+ influx. Levels of Na+ were attenuated in NMDA treated neurons in contrast to untreated neurons. Recently, Keidrowski et al. Pointed out that glutamato-evoked increases in [Na+]i may play a role in Ca2+ homeostasis destabilization. This is consistent with our observations which higher glutamate-evoked levels of [Na+]i in untreated neurons was associated with impaired ability to maintain [Ca2+]i homeostasis. Perhaps this is due to impairment of the ability of these neurons to extrude Ca2+ via the Na+/Ca2+ exchanger. In contrast, NMDA treated neurons had lower glutamate-induced elevation of [Na+]i and enhanced ability to maintain [Ca2+]i level. At present it is not clear whether the glutamate-evoked [Na+]i level is higher in untreated neurons may due to enhanced [Na+]i influx or reduced efflux from the cell. Maintaining ATP levels is essential to maintain an effective transmembrance Na+ gradient. Our data show that cells treated with NMDA have the same basal ATP content as untreated neurons, but are better than untreated neurons to maintain ATP when exposed to glutamate. Lower ATP can impair ATP dependent (i. e. Na+/K+ ATPase) extraction of Na+ from the cells. In addition, lower ATP may reduce activity of endoplasmic Ca2+ ATPase thereby decreasing Ca2+ uptake into intracellular stores and further destabilize Ca2+ homeostasis. This may explain why [Na+]i levels are higher in untreated neurons. It is not understood what mechanism for responsibility to reducing the consumption of ATP content while exposure to glutamate in NMDA-treated neurons. There was an evidence in our previously reported that NMDA receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis[9], suggesting that some transcription steps are involved in the excitoprotection mechanism.
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References
1 Choi, D W. Calcium-mediated neurotoxicity:relationship to specific channel type and role in ischemic damage. Trends Neurosci, 1988,11:465
2 Chuang D, Gao X, Paul S M. N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells. Mol Pharmocol, 1992,42:210
3 Williams. P D, Irwin R P, Lin S Z,et al. Characterization of the excitoprotective actions of N-methyl-D-aspartate in cultured cerebellar granule neurons. J Neurochem, 1995,65:1069
, http://www.100md.com
4 Lin. S Z, Yan G M, Koch. K E, et al. Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores. Brain Res, 1997,771:184
5 Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 1985,260(6):3440
6 Kiedrowski L, Worblewski J T, Costa E. Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate. Mol Pharmacol. 1994,45(5):1050
, 百拇医药
7 Harootunian A C, Kao J P Y, Eckert B K, et al. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J Biol Chem, 1989,264:19458
8 Glinn M, Ni B, Irwin R P,et al. Inorganic Pi increases neuronal survival in the acute early phase following excitotoxic/oxidative insults. J Neurochem, 1998,70:1850
9 Marini A M, Paul S M. N-methyl-D-aspartate receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis. Proc Natl Acad Sci USA, 1992,89:6555
10 Andreeva N, Khodorov B, Stelmashook E, et al. Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures. Brain Research,1991,548:322
(1998 - 09 - 23收稿 1998 - 12 - 08修回), 百拇医药
单位：林穗珍 李晓瑜 中山医科大学附属第一医院儿科；颜光美 药理教研室； 广州， 510080；林穗珍 Robert P. Irwin 颜光美 美国印第安纳大学医学院药理、毒理系； 印第安纳波利斯， 46202)
关键词：谷氨酸；神经元；钙；钠；MK801；兴奋性氨基酸类；N-甲基-天冬氨酸；神经保护药
中山医科大学学报990403Intracellular Ca2+, Na+, and ATP Homeostasis Mediated NMDA-induced Excitoprotection in Rat Cerebellar Granule Neurons
Lin Suizhen1,3 Robert P. Irwin3 Li Xiaoyu1 Yan Guangmei2,3
, 百拇医药
( 1 Department of Pediatrics, First Affiliated Hospital 2 Department of Pharmacology, Sun Yat-sen University of Medical Sciences,Guangzhou, 510089; 3 Department of Pharmacology and Toxicology, Indiana University,School of Medicine, Indianapolis, IN 46202)
Abstract Objective:To study the mechanisms underlying N-methyl-D-aspartate (NMDA)-induced excitoprotection in cultured rat cerebellar granule neurons. Methods: Intracellular Ca2+ ([Ca2+]i) and intracellular Na+([Na+]i) were measured microspectrofluorimetrically with the Ca2+ sensitive dye fura-2/Am and the Na+-sensitive indicator SBFI/AM respectively. Cytosolic denosine triphosphate(ATP) was quantified by means of reverse-phase HPLC. Results: First. Glutamate rapidly induced a peak [Ca2+]i response in both untreated and NMDA treated neurons. In NMDA treated neurons, [Ca2+]i then rapidly dropped to a stable plateau concentration above that of the basal condition. Thirty minutes later, with the removal of glutamate, [Ca2+]i rapidly returned to near basal concentrations. In contrast, with NMDA-untreated neurons, prolonged glutamate exposure resulted in a heterogeneous [Ca2+]i response in many neurons,with [Ca2+]i either gradually or abruptly increasing. Upon glutamate removal, many untreated neurons failed to return to basal levels. Second,Glutamate rapidly induced a peak [Na+]i response in both untreated and NMDA-treated neurons. However, the glutamate-induced [Na+]i peaks, during and after glutamate were all higher in untreated neurons, compared with NMDA-pretreated neurons. Third, Glutamate induced the consumption of ATP stores in the neurons. However,ATP consumption in the neurons pretreated with NMDA was significantly reduced,compared with neurons not pretrect. Conclusions:NMDA-induced excitoprotection in cerebellar granule neurons is associated with enhanced [Ca2+]i and [Na+]i homeostasis by reducing ATP consumption.
, 百拇医药
Subject headings glutamate; neurons; calcium; sodium; MK801; excitatory amino acids, N-methylaspartate; neuroprotective agents
摘 要 目的：研究N-甲基-D-天冬氨酸(NMDA)诱导大鼠小脑颗粒神经元兴奋毒性保护作用的机制。方法：分别用Ca2+和Na+敏感的染料Fura-2/AM和SBFI/AM测定胞内Ca2+和Na+浓度[(Ca2+)i,(Na+)i]。并用反向高效液相色谱法测定胞内三磷酸腺苷(ATP)的含量。结果：①在NMDA预处理组与非处理组，谷氨酸均迅速触发胞内Ca2+反应高峰，两者无显著性差异。而后在NMDA处理组，胞内Ca2+迅速下降，并维持在高于静息Ca2+水平的平台状，撤离谷氨酸后，[Ca2+]i迅速下降并恢复至静息水平；而NMDA非处理组则相反。②谷氨酸均诱发上述两组神经元[Na+]i升高，在NMDA非处理组，[Na+]i均高于NMDA预处理组。③谷氨酸消耗胞内ATP， 而NMDA预处理组则减少胞内ATP的耗竭。结论：NMDA诱导兴奋毒性保护作用是通过减少ATP的消耗而增强胞内Ca2+和Na+的自稳态。
, 百拇医药
中图号 R 979
Glutamate, the major excitatory neurotransmitter, is involved in many important CNS functions. However, it can also be an excitotoxin and is thought to be involved in the pathogenesis of neuronal death due to cerebral ischemia and trauma. Application of toxic concentrations of glutamate to cerebellar granule neurons can produce excessive increases in intracellular calcium ([Ca2+]i) and results in cell death[1]. Over stimulation of both N-methyl-D-aspartate (NMDA) and non-NMDA receptor subtypes may be involved in the excitotoxicity of glutamate. Exposure of cultured rat cerebellar granule neurons to subtoxic NMDA concentration has previously been shown to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations demonstrated by reduced susceptibility to glutamate[2]. Also,we showed that subtoxic concentrations of NMDA could shift the concentration-response for glutamate toxicity in a concentration and time-dependent manner. This effect has been shown to be dependent on protein synthesis[3]. In this study we have further characterized intracellular excitoprotection in cerebellar granule neurons including: changes in intracellular calcium([Ca2+]i), intracellular sodium ([Na+]i) homeostasis and alteration of the energy metabolic state.
, 百拇医药
1 Materials and Methods
1.1 Materials
L-Glutamate, NMDA, and MK-801 were from RBI. Fura-2/AM and SBFI/AM were from molecular probes. Ionomycin was from calbiochem. Ethylene imine polymer was from fluka chemical corp. All other chemicals were purchased from Sigma.
1.2 Primary neurons culture
Rat cerebellar granule neurons were prepared from 8-day-old Sprague-Dawley rat pups as described previously[3]. Briefly, neurons were dissociated from freshly dissected cerebellar by mechanical disruption in the presence of trypsin and DNase, and then plated in poly-1-lysine-coated dishes. Cells were seeded at a density of (1.5～1.8)×106 cells/mL in basal modified Eagle′s medium (BME) containig 10% fetal bovine serum and 25 mmol/L KCl. Cytosine arabinoside (10 μmol/L) was added to the culture medium after 24h to arrest the growth of non-neuronal cells. D-Glucose (5 mmol/L) was added to the cultures on day 7 and every fourth day thereafter.
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1.3 Measurement of Intracellular Ca2+
Measurement of [Ca2+]i was carried out as previously described using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2[4]. In brief, NMDA (50 μmol/L) was added to the culture medium of pretreated neurons for 24 h. Neurons were incubation with 5 μmol/L Fura-2 for 45 min. Cells were then washed twice with BME containing 25 mmol/L KCl and 10 μmol/L glycine. The cells were continuously perfused with same buffer at 37 ℃. Following that [Ca2+]i was measured for 5 min as a basal [Ca2+]i of neurons, glutamate then was perfused for 30 min. The same buffer was then used to perfuse for another 30 min to wash out glutamate. Neurons were illuminated on an inverted microscope (Nikon Diaphot). Excitation of fura-2 was at 340 and 380 nm. Cell images were digitized and stored for subsequent analysis (Universal Imaging Co, USA). Calculation of [Ca2+]i was carried out as described by Grynkiewicz et al[5] .
, 百拇医药
1.4 Measurement of Intracellular Na+
Measurement of [Na+]i was carried out as previously described using microspectrofluorimetry and the Na+ sensitive indicator SBFI/AM[6]. Briefly, cells was loaded with SBFI/AM(10 μmol/L) and returned to the incubator for 45 min. Cells were washed 3 times and allowed ≥15 min to complete the hydrolysis of the ester linkage before starting imaging. Cells were imagined using identical conditions to that for fura-2 experiments with excitation wavelengths, 340 nm and 380 nm and emitted light monitored at 510 nm. Background intensity levels were subtracted and in situ calibrations were carried out as descried by Harootunian et al[7] .
, 百拇医药
1.5 Measurement of ATP
Untreated and NMDA-treated neurons were exposed to glutamate (100 μmol/L) for 30 min. Cytosolic contents were then extracted with HCl and adenosine phosphonucleotide ([ATP]) levels were quantified with RP-HPLC before exposure to glutamate, following glutamate and removal glutamate[8].
1.6 Statistical Analysis
Data are presented as
±s. Unless otherwise stated, statistical comparisons were made using studentt-test., 百拇医药
2 Results
2.1 NMDA-induced excitoprotection enhances [Ca2+]i homeostasis
Since glutamate-induced cell death is related to increase in [Ca2+]i[1], we measured changes in [Ca2+]i associated with a 30 min exposure to glutamate in NMDA-pretreated and untreated cerebellar granule neurons in cultures. In NMDA-pretreatment of granule neurons resulted in a lessees reduction in basal [Ca2+]i but glutamate rapidly induced a peak [Ca2+]i response in both untreated and NMDA treated neurons. In NMDA treated neurons, [Ca2+]i then rapidly lowered to a stable plateau concentration above that of the basal condition. Thirty minutes later with the removal of glutamate,[Ca2+]i rapidly returned to near basal concentrations. In contrast, in case of untreated neurons prolonged glutamate exposure resulted in a heterogeneous [Ca2+]i response with many neurons gradually or abruptly increased [Ca2+]i. Upon removal of glutamate many untreated neurons failed to return to basal levels (Fig 1).
, http://www.100md.com
Fig.1 NMDA-induced excitoprotection enhances [Ca2+]i homeostasis
[Ca2+]i was measured with the fluorescent Ca2+ indicator Fura-2. A, following recording basal [Ca2+]i, glutamate 100 μmol/L was applied for 30 min to cerebellar granule neurons pretreated for 24 h with NMDA (50 μmol/L). The experiment was terminated 30 min following application of glutamate. Each line (-) represent the calculted [Ca2+]i concentration of one cerebellar granule neuron.B, as in(A) but cerebellar granule neurons were untreated with NMDA (n =30 in each condition)
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To assess the role of the glutamate receptor subtypes in the NMDA-induced changes in Ca2+ homeostasis, we used the noncompetitive NMDA antagonist MK-801 to block the NMDA component of the glutamate-induced rise in [Ca2+]i. Experiments were performed as those with glutamate exposure alone except MK-801 (1 μmol/L) was perfused over the neurons for 2 min before addition of glutamate. MK-801 markedly reduced the glutamate-induced rise in [Ca2+]i and eliminated the differences responce between untreated and NMDA pretreated neurons in the 30 min during and after glutamate exposure.
, 百拇医药
2.2 NMDA-induced excitoprotection enhances [Na+]i homeostasis
To examine the effects of NMDA pretreatment on intracellular Na+ ([Na+]i) homeostasis, we measured [Na+]i using the Na+ sensitive indicator SBFI/AM.[Na+]i of cerebellar granule neurons was monitored before, during and following exposure to 100 μmol/L glutamate. Glutamate-induced a rapid elevation of [Na+]i from basal levels(Fig 2).
, 百拇医药
Fig. 2 NMDA-pretreatment of cerebellar granule neurons attenuates glutamateinduced eleva-
tion of intracellular Na+
c([Na+]i) was measured with the fluorescent Na+ indicator SBFI/AM. The drug application as described in Fig1. Straight-line (-) represent the mean of calculated [Na+]i concentration of NMDA-untreated cerebellar granule neurons. Circle line (-o-) represent the mean of calculated [Na+]i concentration of preexposed to NMDA (50 μmol/L) for 24 h (n =161 in each contion)
, 百拇医药
In both untreated and NMDA pretreated neurons, glutamate-induced a stable plateau concentration of [Na+]i above that of the basal condition. However, the magnitude of the response differed greatly between these groups. The glutamate-induced [Na+]i peak, during glutamate and the post glutamate (30 min) were both greater in the untreated than in the NMDA pretreated neurons.
2.3 NMDA-pretreatment effects on glutamate-induced changes of ATP content
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ATP content was determined on cerebellar granule neurons before glutamate, following a 30 min exposure to glutamate (100 μmol/L) and 30 min following the washout of glutamate. NMDA treatment did not alter the mean ATP (% of control) content. Exposure to glutamate induced a consumption of ATP store. However, pretreatment with NMDA reduced the consumption of ATP (18% difference) than observed in untreated cells. Even a larger degradation (28% difference) follows a 30 min recovery period in untreated cells (Fig 3).
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Fig.3 NMDA-pretreatment of granule neurons reducedglutamate-induced depletion of ATP content
Data are from a representative experiment repeated three times with qualitatively similar results. Each point represents a mean of four replicates.*Statistically significant difference (P<0.05) between. NMDA-pretreated and untreated
3 Discussion
In the present study we have extended our previous ly findings that preincubation of cultured cerebellar granule neurons with subtoxic concentrations of NMDA for 24 h results in the induction of exitoprotective state as measured by subsequent exposure to glutamate. We found that NMDA pretreatment-induced alterations of Ca2+ and Na+ homeostasis in cerebellar granule neurons. Since MK-801 can completely block glutamate toxicity at the concentrations used in this study[3], could the NMDA-induced alterations of Ca2+ homeostasis in cerebellar granule neurons be simply due to “down-regulation” of NMDA receptors? We previous reported that exposure of NMDA results in no change of specific [3H] MK-801 binding to NMDA receptors and the immediate peak response in [Ca2+]i induced by glutamate was unaltered in cerebellar granule neurons preexposed to NMDA. Additionally, NMDA receptor-mediated neuroprotection extends to MPP+ toxicity in cerebellar granule neurons[9]. It is suggested that the NMDA-induced excitoprotection and maintain of Ca2+ homeostasis in cerebellar granule neurons is not due to “down-regulation” of NMDA receptors. Under both basal and pathological conditions, intracellular free Ca2+ concentration is stringently controlled because excessive Ca2+ influx is lethal to neurons. The transmembrane Na+ gradient is also tightly regulated; excessive Na+ influx which occurs under excitotoxic or ischemic conditions, may lead to inordinate excitatory amino acid efflux, as well as osmotic imbalance, cell swelling, and lysis[10]. Our data show that glutamate-induced influx of Na+ into the cerebellar granule neurons both in NMDA treated and untreated neurons, similar pattern was observed with Ca2+ influx. Levels of Na+ were attenuated in NMDA treated neurons in contrast to untreated neurons. Recently, Keidrowski et al. Pointed out that glutamato-evoked increases in [Na+]i may play a role in Ca2+ homeostasis destabilization. This is consistent with our observations which higher glutamate-evoked levels of [Na+]i in untreated neurons was associated with impaired ability to maintain [Ca2+]i homeostasis. Perhaps this is due to impairment of the ability of these neurons to extrude Ca2+ via the Na+/Ca2+ exchanger. In contrast, NMDA treated neurons had lower glutamate-induced elevation of [Na+]i and enhanced ability to maintain [Ca2+]i level. At present it is not clear whether the glutamate-evoked [Na+]i level is higher in untreated neurons may due to enhanced [Na+]i influx or reduced efflux from the cell. Maintaining ATP levels is essential to maintain an effective transmembrance Na+ gradient. Our data show that cells treated with NMDA have the same basal ATP content as untreated neurons, but are better than untreated neurons to maintain ATP when exposed to glutamate. Lower ATP can impair ATP dependent (i. e. Na+/K+ ATPase) extraction of Na+ from the cells. In addition, lower ATP may reduce activity of endoplasmic Ca2+ ATPase thereby decreasing Ca2+ uptake into intracellular stores and further destabilize Ca2+ homeostasis. This may explain why [Na+]i levels are higher in untreated neurons. It is not understood what mechanism for responsibility to reducing the consumption of ATP content while exposure to glutamate in NMDA-treated neurons. There was an evidence in our previously reported that NMDA receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis[9], suggesting that some transcription steps are involved in the excitoprotection mechanism.
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