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Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

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Abstract

1.Previous studies demonstrated that estrogens, specifically 17β-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17β-estradiol and its weaker isomer, 17α-estradiol, in both anoxia-reoxygenation and glutamate toxicity.

2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.

3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17β-estradiol (1.3 or 133 nM) and 17α-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17β-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17α-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.

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REFERENCES

  • Alkayed, N. J., Harukuni, I., Kimes, A. S., London, E. D., Traystman, R. J., and Hurn, P. D. (1998). Gender-linked brain injury in experimental stroke. Stroke 29:159–165.

    Google Scholar 

  • Azbill, R. D., Mu, X., Bruce-Keller, A. J., Mattson, M. P., and Springer, J. E. (1997). Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res. 765:283–290.

    Google Scholar 

  • Behl, C., Widmann, M., Trapp, T., and Holsboer, F. (1995). 17-β Estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem. Biophys. Res. Commun. 216:473–482.

    Google Scholar 

  • Behl, C., Skutella, T., Lezoualc'h, F., Post, A., Widmann, M, Newton, C. J., and Holsboer, F. (1997). Neuroprotection against oxidative stress by estrogens: structure activity relationship. Mol. Pharmacol. 51:535–541.

    Google Scholar 

  • Bengtsson, F., and Siesjo, B. K. (1989). Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: A unifying hypothesis. J. Cereb. Blood Flow Metab. 9:127–140.

    Google Scholar 

  • Benveniste, H., Drejer, J., Schousboe, A., and Diemer, N. H. (1984). Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43:1369–1374.

    Google Scholar 

  • Bishop, J., and Simpkins, J. W. (1994). Estradiol treatment increases viability of glioma and neuroblastoma cells in vitro. Mol. Cell. Neurosci. 5:303–308.

    Google Scholar 

  • Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.

    Google Scholar 

  • Braughler, J. M., and Hall, E. D. (1989). Central nervous system trauma and stroke. I. biochemical considerations for oxygen radical formation and lipid peroxidation. Free Rad. Bio. Med. 6:289–301.

    Google Scholar 

  • Brinton, R. D. (1993). 17β-Estradiol induction of filopodial growth in cultured hippocampal neurons within minutes of exposure. Mol. Cell Neurosci. 4:36–46.

    Google Scholar 

  • Brinton, R. D., Proffitt, P., Tran, J., and Luu, R. (1997a). Equilin, a principal component of the estrogen replacement therapy premarin, increases the growth of cortical neurons via an NMDA receptor-dependent mechanism. Exp. Neurol. 147:211–220.

    Google Scholar 

  • Brinton, R. D., Tran, J., Proffitt, P., and Kahil, M. (1997b). 17β-Estradiol enhances the outgrowth and survival of neocortical neurons in culture. Neurochem. Res. 22:1339–1351.

    Google Scholar 

  • Buchan, A. M., Xue, D., Huang, Z. G., Smith, K. H., and Lesiuk, H. (1991). Delayed AMPA receptor blockade reduces cerebral infarction induced by focal ischemia. Neuroreport. 2:473–476.

    Google Scholar 

  • Bullock, R. (1995). Strategies for neuroprotection with glutamate antagonists. Extrapolating form evidence taken from the first stroke and head injury studies. Ann. N.Y. Acad. Sci. 765:272–278.

    Google Scholar 

  • Chandler, L. J., Sumners, C., and Crews, F. T. (1993). Ethanol inhibits NMDA receptor-mediated excitotoxicity in rat primary neuronal cultures. Alcohol Clin. Exp. Res. 17:54–60.

    Google Scholar 

  • Choi, D. W. (1987). Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7:369–379.

    Google Scholar 

  • Choi, D. W. (1988). Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634.

    Google Scholar 

  • Choi, D. W. (1992). Excitotoxic cell death. J. Neurobiol. 23:1261–1276.

    Google Scholar 

  • Clark, J. H., and Markaverich, B. M. (1983). The agonistic and antagonistic effects of short acting estrogens: A review. Pharmacol. Ther. 21:429–453.

    Google Scholar 

  • Clark, J. H., Williams, M., Upchurch, S., Eriksson, H., Helton, E., and Markaverich, B. M. (1982). Effects of estradiol-17 alpha on nuclear occupancy of the estrogen receptor, stimulation of nuclear type II sites and uterine growth. J. Steroid Biochem. 16:323–328.

    Google Scholar 

  • Davis, J. B., and Maher, P. (1994). Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res. 652:169–173.

    Google Scholar 

  • Dubal, D. B., Kashon, M. L., Pettigrew, L. C., Ren, J. M., Finklestein, S. P., Rau, S. W., and Wise, P. M. (1998). Estradiol protects against ischemic injury. J. Cereb. Blood Flow 18:1253–1258.

    Google Scholar 

  • Dugan, L. L., and Choi, D. W. (1994). Excitotoxicity, free radicals, and cell membrane changes. Ann. Neurol. 35:17–21.

    Google Scholar 

  • Fillit, H., Weinreb, H., Cholst, I., Luine, V., McEwen, B., Amador, R., and Zabriskie, J. (1985). Observations in a preliminary open trial of estradiol therapy for senile dementia-Alzheimer's type. Psychoneuroendocrinology 11:337–345.

    Google Scholar 

  • Finucane, F. F., Madans, J. H., Bush, T. L., Wolf, P. H., and Kleinman, J. C. (1993). Decreased risk of stroke among postmenopausal hormone users. Results from a national cohort. Arch. Intern. Med. 153:73–79.

    Google Scholar 

  • Frandsen, A., Drejer, J., and Schousboe, A. (1989). Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-D-aspartate (NMDA) as well as non-NMDA receptors. J. Neurochem. 53:297–299.

    Google Scholar 

  • Gasic, G. P., and Hollmann, M. (1992). Molecular neurobiology of glutamate receptors. Annu. Rev. Physiol. 54:507–536.

    Google Scholar 

  • Goldberg, M. P., Weiss, J. H., Phuong-Chi, P., and Choi, D. W. (1987). N-Methyl D-aspartate receptors mediate hypoxic neuronal injury in cortical culture. J. Pharmacol. Exp. Ther. 243:784–791.

    Google Scholar 

  • Goodman, Y., Bruce, A. J., Cheng, B., and Mattson, M. P. (1996). Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid β-peptide toxicity in hippocampal neurons. J. Neurochem. 66:1836–1844.

    Google Scholar 

  • Green, P. S., Gridley, K. E., and Simpkins, J. W. (1996). Estradiol protects against β-amyloid (25–35)-induced toxicity in SK-N-SH human neuroblastoma cells. Neurosci. Lett. 218:165–168.

    Google Scholar 

  • Green, P. S., Bishop, J., and Simpkins, J. W. (1997a). 17 α-Estradiol exerts neuroprotective effects on SK-N-SH cells. J. Neurosci. 17:511–515.

    Google Scholar 

  • Green, P. S., Gordon, K., and Simpkins, J. W. (1997b). Phenolic A ring requirement for the neuroprotective effects of steroids. J. Steroid Biochem. Mol. Biol. 63:229–235.

    Google Scholar 

  • Green, P. S., Gridley, K. E., and Simpkins, J. W. (1998). Nuclear estrogen receptor-independent neuroprotection by estratrienes: A novel interaction with glutathione. Neuroscience 84:7–10.

    Google Scholar 

  • Gridley, K. E., Green, P. S., and Simpkins, J. W. (1997). Low concentrations of estradiol reduce β-amyloid (25–35)-induced toxicity, lipid peroxidation and glucose utilization in human SK-N-SH neuroblastoma cells. Brain Res. 778:158–165.

    Google Scholar 

  • Hagberg, H., Lehmann, A., Sandberg, M., Nystrom, B., Jacobson, I., and Hamberger, A. (1985). Ischemiainduced shift of inhibitory and excitatory amino acids from intra-to extracellular compartments. J. Cereb. Blood Flow Metab. 5:413–419.

    Google Scholar 

  • Hartikka, J., and Hefti, F. (1988). Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes. J. Neurosci. 8:2967–2985.

    Google Scholar 

  • Hastings, T. G., and Reynolds, I. J. (1995). Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J. Neurosci. 15:3318–3327.

    Google Scholar 

  • Henderson, V. W., Paganini-Hill, A., Emanuel, C. K., Dunn, M. E., and Buckwalter, J. G. (1994). Estrogen replacement therapy in older women. Comparisons between Alzheimer's disease cases and nondemented control subjects. Arch. Neurol. 51:896–900.

    Google Scholar 

  • Huggins, C., Jensen, E. V., and Cleveland, A. S. (1954). Chemical structure of steroids in relation to promotion of growth of the vagina and uterus of the hypophysectomized rat. J. Exp. Med. 100:225–243.

    Google Scholar 

  • Kneifel, M. A., Leytus, S. P., Fletcher, E., Weber, T., Mangel, W. F., and Katzenellenbogen, B. S. (1982). Uterine plasminogen activator activity: modulation by steroid hormones. Endocrinology 111:493–499.

    Google Scholar 

  • Korenman, S. G. (1969). Comparative binding affinity of estrogens and its relation to estrogenic potency. Steroids 13:163–177.

    Google Scholar 

  • Kuiper, G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., and Gustafsson, J. A. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology 138:863–870.

    Google Scholar 

  • Lacort, M., Leal, A. M., Liza, M., Martin, C., Martinez, R., and Ruiz-Larrea, M. B. (1995). Protective effects of estrogens and catecholestrogens against peroxidative membrane damage in vitro. Lipids 30:141–146.

    Google Scholar 

  • Lipton, S. A., and Rosenberg, P. A. (1994). Excitatory amino acids as a final common pathway for neurologic disorders. N. Engl. J. Med. 330:613–622.

    Google Scholar 

  • Lubahan, D. B., McCarty, K. S., Jr., and McCarty, K. S., Sr. (1985). Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors. J. Biol. Chem. 260:2515–2526.

    Google Scholar 

  • Michaels, R. L., and Rothman, S. M. (1990). Glutamate neurotoxicity in vitro: Antagonist pharmacology and intracellular calcium concentrations. J. Neurosci. 10:283–292.

    Google Scholar 

  • Morley, P., Hogan, M. J., and Hakim, A. M. (1994). Calcium-mediated mechanisms of ischemic injury and protection. Brain Pathol. 4:37–47.

    Google Scholar 

  • Murphy, T. H., Miyamoto, M., Sastre, A., Schnaar, R. L., and Coyle, J. T. (1989). Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2:1547–1558.

    Google Scholar 

  • Nakano, M., Sugioka, K., Naito, I., Takekoshi, S., and Niki, E. (1987). Novel and potent biological antioxidants on membrane phospholipid peroxidation: 2-Hydroxyestrone and 2-hydroxyestradiol. Biochem. Biophys. Res. Commun. 142:919–924.

    Google Scholar 

  • Ohkura, T., Isse, K., Akazawa, K., Hamamoto, M., Yaoi, Y., and Hagino, N. (1994). Low-dose estrogen replacement therapy for Alzheimer disease in women. Menopause 1:125–130.

    Google Scholar 

  • Ohkura, T., Isse, K., Akazawa, K., Hamamoto, M., Yaoi, Y., and Hagino, N. (1995). Long-term estrogen replacement therapy in female patients with dementia of the Alzheimer's type 7 case reports. Dementia 6:99–107.

    Google Scholar 

  • Olney, J. W. (1986). Inciting excitotoxic cytocide among central neurons. Adv. Exp. Med. Biol. 203:631–645.

    Google Scholar 

  • O'Malley, E. K., Black, I. B., and Dryfus, C. F. (1991). Local support cells promote survival of substantia nigra dopaminergic neurons in culture. Exp. Neurol. 112:40–48.

    Google Scholar 

  • Paganini-Hill, A. (1995). Estrogen replacement therapy and stroke. Prog. Cardiovasc. Dis. 38:223–242.

    Google Scholar 

  • Park, C. K., Nehls, D. G., Graham, D. I., Teasdale, G. M., and McCulloch, J. (1988). The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat. Ann. Neurol. 24:543–551.

    Google Scholar 

  • Shi, J., Zhang, Y. Q., and Simpkins, J. W. (1997). Effects of 17β-estradiol on glucose transporter 1 expression and endothelial cell survival following focal ischemia in the rats. Exp. Brain Res. 117:200–206.

    Google Scholar 

  • Siesjo, B. K., Zhao, Q., Pahlmark, K., Seisjo, P., Katsura, K., and Folbergrova, J. (1995). Glutamate, calcium, and free radicals as mediators of ischemic brain damage. Ann. Thorac Surg. 59:1316–1320.

    Google Scholar 

  • Simpkins, J. W., Rajakumar, G., Zhang, Y. Q., Simpkins, C. E., Greenwald, D., Yu, C. J., Bodor, N., and Day, A. L. (1997). Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J. Neurosurg. 87:724–730.

    Google Scholar 

  • Singer, C. A., Rogers, K. L., Strickland, T. M., and Dorsa, D. M. (1996). Estrogen protects primary cortical neurons from glutamate toxicity. Neurosci. Lett. 212:13–16.

    Google Scholar 

  • Speicher, D. W., Peace, J. N., and McCarl, R. L. (1981). Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures. In Vitro 17:863–870.

    Google Scholar 

  • Sugioka, K., Shimosegawa, Y., and Nakano, M. (1987). Estrogens as natural antioxidants of membrane phospholipid peroxidation. FEBS Lett. 210:37–39.

    Google Scholar 

  • Takanashi, K., Watanabe, K., and Yoshizawa, I. (1995). On the inhibitory effects of C17-sulfoconjugated catechol estrogens upon lipid peroxidation of rat liver microsomes. Biol. Pharm. Bull. 18:1120–1125.

    Google Scholar 

  • Tang, M., Abplanalp, W., Ayres, S., and Subbiah, M. T. (1996). Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation. Metabolism 45:411–414.

    Google Scholar 

  • Velazquez, J. L., Frantseva, M. V., and Carlen, P. L. (1997). In vitro ischemia promotes glutamatemediated free radial generation and intracellular calcium accumulation in hippocampal pyramidal neurons. J. Neurosci. 17:9085–9094.

    Google Scholar 

  • Weaver, C. E., Jr., Park-Chung, M., Gibbs, T. T., and Farb, D. H. (1997). 17β-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors. Brain Res. 761:338–341.

    Google Scholar 

  • Wong, M., and Moss, R. L. (1992). Long-term and short-term electrophysiological effects of estrogen on the synaptic properties of hippocampal CA1 neurons. J. Neurosci. 12:3217–3225.

    Google Scholar 

  • Wren, B. G. (1992). The effect of oestrogen on the female cardiovascular system. Med. J. Aust. 157:204–208.

    Google Scholar 

  • Zhang, Y. Q., Shi, J., Rajakumar, G., Day, A. L., and Simpkins, J. W. (1998). Effects of gender and estradiol treatment on focal brain ischemia. Brain Res. 784:321–324.

    Google Scholar 

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Zaulyanov, L.L., Green, P.S. & Simpkins, J.W. Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens. Cell Mol Neurobiol 19, 705–718 (1999). https://doi.org/10.1023/A:1006948921855

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