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The Difference Between The Response To Glutamate Excitotoxicity and The Role Of Ca2+ Channel Blockers in Cortical Neuron and SH-SY5Y Cells Cultures

Year 2022, Volume: 7 Issue: 1, 45 - 52, 31.03.2022
https://doi.org/10.35229/jaes.1003400

Abstract

Cortical neuron and SH-SY5Y cells are widely used in glutamate excitotoxicity studies, but it is unclear which one better reflects this model. Generally, glutamate induces toxicity conditions by leading to L and L/N-Ca2+channels activation and cell death via lethal Ca2+ influx. To evaluate this hypothesis, the effects of L and L/N-Ca2+ channel blockers, lacidipine, and amlodipine under excitotoxic conditions were evaluated. At the same time, in this study, we aimed to determine that these two cell lines better reflect this model. To induce excitotoxicity, cortical neuron and SH-SY5Y cells were incubated with glutamate 10-5 mM. After 30 min incubation with glutamate, different concentration (1, 2 and 4 µg lacidipine and 20, 50 and 100 µM amlodipine) were applied these cells. Possible neuroprotective roles of lacidipine and amlodipine were investigated through cell viability, oxidative stress, and apoptotic alterations. Our results showed that SH-SY5Y cells are the more ideal cell line for oxidative stress-mediated glutamate toxicity. In addition, 4 µg lacidipine and 100 µM amlodipine had significant neuroprotective roles in these cells, but the most protective effect was also determined in SH-SY5Y cells at 100 µM amlodipine. The highest viability rate on cell lines was found at 88,8 % in SH-SY5Y cells treated with 100 μM amlodipine. Results from the TAC, TOS, LDH assays, and flow cytometry analysis were correlated to our MTT results. Taken together, our results indicate that SH-SY5Y cells are more effective at reflecting glutamate-induced excitotoxicity and 100μM amlodipine has a more protective effect in treating this toxicity.

References

  • Barbosa, D. J., Capela, J. P., de Lourdes Bastos, M., & Carvalho, F. (2015). In vitro models for neurotoxicology research. Toxicology Research, 4(4), 801-842.
  • Binvignat, O., & Olloquequi, J. (2020). Excitotoxicity as a target against neurodegenerative processes. Current Pharmaceutical Design, 26(12), 1251-1262.
  • Carvajal, F. J., Mattison, H. A., & Cerpa, W. (2016). Role of NMDA receptor-mediated glutamatergic signaling in chronic and acute neuropathologies. Neural Plasticity, 1-21.
  • Chávez-Castillo, M., Rojas, M., & Bautista, J. (2017). Excitotoxicity: an organized crime at the cellular level. Archivos de Medicina, 8(3), 198-203.
  • Choi, N. Y., Choi, H., Park, H. H., Lee, E. H., Yu, H. J., Lee, K. Y., & Koh, S. H. (2014). Neuroprotective effects of amlodipine besylate and benidipine hydrochloride on oxidative stress-injured neural stem cells. Brain Research, 1551, 1-12.
  • Dong, X. X., Wang, Y., & Qin, Z. H. (2009). Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacologica Sinica, 30(4), 379-387.
  • Erel, O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical Biochemistry, 37(4), 277-285.
  • Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103-1111.
  • Giansante, G., Marte, A., Romei, A., Prestigio, C., Onofri, F., Benfenati, F., & Valente, P. (2020). Presynaptic L-type Ca2+ channels increase glutamate release probability and excitatory strength in the hippocampus during chronic neuroinflammation. Journal of Neuroscience, 40(36), 6825-6841.
  • Godfraind, T. (2017). Discovery and development of calcium channel blockers. Frontiers in Pharmacology, 8, 286-301.
  • Godoy, J. A., Rios, J. A., Picón-Pagès, P., Herrera-Fernández, V., Swaby, B., Crepin, G., & Muñoz, F. J. (2021). Mitostasis, Calcium and Free Radicals in Health, Aging and Neurodegeneration. Biomolecules, 11(7), 1012-1022.
  • Higley, M. J., & Sabatini, B. L. (2012). Calcium signaling in dendritic spines. Cold Spring Harbor Perspectives in Biology, 4(4), a005686.
  • Hu, Y., Li, J., Liu, P., Chen, X., Guo, D. H., Li, Q. S., & Rahman, K. (2012). Protection of SH-SY5Y neuronal cells from glutamate-induced apoptosis by 3, 6′-disinapoyl sucrose, a bioactive compound isolated from Radix Polygala. Journal of Biomedicine and Biotechnology, 38, 1-5.
  • Khurana, K., & Bansal, N. (2019). Lacidipine attenuates reserpine-induced depression-like behavior and oxido-nitrosative stress in mice. Naunyn-Schmiedeberg's Archives of Pharmacology, 392(10), 1265-1275.
  • Khurana, K., Kumar, M., & Bansal, N. (2021). Lacidipine Prevents Scopolamine-Induced Memory Impairment by Reducing Brain Oxido-nitrosative Stress in Mice. Neurotoxicity Research, 39, 1-16.
  • Kim, T. Y., Niimi, K., & Takahashi, E. (2016). Role of Cav2. 1 Channel Signaling in Glutamate-Related Brain Injury. Brain Disorder Therapy, 5(226), 2-22.
  • Kim, Y. H., Eom, J. W., & Koh, J. Y. (2020). Mechanism of zinc excitotoxicity: a focus on AMPK. Frontiers in Neuroscience, 14, 958-966.
  • Kimura, M., Katayama, K., & Nishizawa, Y. (1999). Role of glutamate receptors and voltage-dependent calcium channels in glutamate toxicity in energy-compromised cortical neurons. The Japanese Journal of Pharmacology, 80(4), 351-358.
  • Krasil’Nikova, I., Surin, A., Sorokina, E., Fisenko, A., Boyarkin, D., Balyasin, M., & Pinelis, V. (2019). Insulin protects cortical neurons against glutamate excitotoxicity. Frontiers in Neuroscience, 13, 1027-1035.
  • Kritis, A. A., Stamoula, E. G., Paniskaki, K. A., & Vavilis, T. D. (2015). Researching glutamate–induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Frontiers in Cellular Neuroscience, 9, 91-103.
  • Lee, Y. M., He, W., & Liou, Y. C. (2021). The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide. Cell Death & Disease, 12(1), 1-13.
  • Mahajan, A. S., Babbar, R., Kansal, N., Agarwal, S. K., & Ray, P. C. (2007). Antihypertensive and antioxidant action of amlodipine and vitamin C in patients of essential hypertension. Journal of Clinical Biochemistry and Nutrition, 40(2), 141-147.
  • Manev, H., Kharlamov, E., Uz, T., Mason, R. P., & Cagnoli, C. M. (1997). Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells. Experimental Neurology, 146(1), 171-178.
  • Sanchez, A. B., Medders, K. E., Maung, R., Sánchez-Pavón, P., Ojeda-Juárez, D., & Kaul, M. (2016). CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l-type Ca 2+ channels upstream of p38 MAPK. Journal of Neuroinflammation, 13(1), 1-12.
  • Stuiver, B. T., Douma, B. R. K., Bakker, R., Nyakas, C., & Luiten, P. G. M. (1996). In vivo protection against NMDA-induced neurodegeneration by MK-801 and nimodipine: combined therapy and temporal course of protection. Neurodegeneration, 5(2), 153-159.
  • Sun, Z. W., Zhang, L., Zhu, S. J., Chen, W. C., & Mei, B. (2010). Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage. Neuroscience Bulletin, 26(1), 8-16.
  • Taghizadehghalehjoughi, A., & Naldan, M. E. (2018). The study of diazepam, pregabalin and glucose effect on glutamate toxicity: In vitro study. Medicine, 7(4), 797-801.
  • Vallazza-Deschamps, G., Fuchs, C., Cia, D., Tessier, L. H., Sahel, J. A., Dreyfus, H., & Picaud, S. (2005). Diltiazem-induced neuroprotection in glutamate excitotoxicity and ischemic insult of retinal neurons. Documenta Ophthalmologica, 110(1), 25-35.
  • Valtcheva, S., & Venance, L. (2019). Control of long-term plasticity by glutamate transporters. Frontiers in Synaptic Neuroscience, 11, 10-22.
  • Wheeler, D. B., Randall, A., & Tsien, R. W. (1996). Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca2+ channels in rat hippocampus. Journal of Neuroscience, 16(7), 2226-2237.
  • Yang, X. R., Sun, P., Qin, H. P., Si, P. P., Sun, X. F., & Zhang, C. (2012). Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons. Acta physiologica Sinica, 64(6), 609-616.
  • Zyśk, M., Gapys, B., Ronowska, A., Gul-Hinc, S., Erlandsson, A., Iwanicki, A.,& Bielarczyk, H. (2018). Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells. PLoS One, 13(12), e0209363.

Kortikal Nöron ve SH-SY5Y Hücre Kültürlerinde Glutamat Eksitotoksisitesine Yanıt ile Ca2+ Kanal Blokerlerinin Rolü Arasındaki Fark

Year 2022, Volume: 7 Issue: 1, 45 - 52, 31.03.2022
https://doi.org/10.35229/jaes.1003400

Abstract

Kortikal nöron ve SH-SY5Y hücreleri glutamat eksitotoksisite çalışmalarında yaygın olarak kullanılmaktadır, ancak hangisinin bu modeli daha iyi yansıttığı belirsizdir. Genel olarak glutamat, L ve L/N-Ca2+ kanallarının aktivasyonuna ve öldürücü Ca2+ akışı yoluyla hücre ölümüne yol açarak toksisite koşullarını indükler. Bu hipotezi değerlendirmek için eksitotoksik koşullar altında L ve L/N-Ca2+ kanal blokerleri, lasidipin ve amlodipinin etkileri değerlendirildi. Aynı zamanda bu çalışmada bu iki hücre hattının bu modeli daha iyi yansıttığını belirlemeyi amaçladık. Eksitotoksisiteyi indüklemek için kortikal nöron ve SH-SY5Y hücreleri glutamat 10-5 mM mM ile inkübe edildi. Glutamat ile 30 dakikalık inkübasyondan sonra, bu hücrelere farklı konsantrasyonlar (1, 2 ve 4 µg lasidipin ve 20, 50 ve 100 μM amlodipin) uygulandı.Lasidipin ve amlodipinin olası nöroprotektif rolleri, hücre canlılığı, oksidatif stres ve apoptotik değişiklikler yoluyla araştırıldı. Sonuçlarımız SH-SY5Y hücrelerinin oksidatif stres aracılı glutamat toksisitesini daha iyi yansıttığını gösterdi. 4 µg lasidipin ve 100 μM amlodipin bu hücrelerde önemli nöroprotektif rollere sahip olmasına rağmen en yüksek koruyucu etki 100 μM amlodipin'de SH-SY5Y hücrelerinde de belirlendi. En yüksek canlılık oranı, 100 μM amlodipin ile tedavi edilen SH-SY5Y hücrelerinde %88,8 olarak bulundu.TAC, TOS, LDH tahlilleri ve flow sitometrik analizinden elde edilen sonuçlar MTT sonuçlarımızla benzerdi. Tüm sonuçlar birlikte değerlendirildiğinde SH-SY5Y hücrelerinin glutamat kaynaklı eksitotoksisiteyi yansıtmada daha etkili hücre hattı olduğunu ve 100 μM amlodipinin bu toksisitenin tedavisinde daha koruyucu bir etkiye sahip olduğunu göstermektedir.

References

  • Barbosa, D. J., Capela, J. P., de Lourdes Bastos, M., & Carvalho, F. (2015). In vitro models for neurotoxicology research. Toxicology Research, 4(4), 801-842.
  • Binvignat, O., & Olloquequi, J. (2020). Excitotoxicity as a target against neurodegenerative processes. Current Pharmaceutical Design, 26(12), 1251-1262.
  • Carvajal, F. J., Mattison, H. A., & Cerpa, W. (2016). Role of NMDA receptor-mediated glutamatergic signaling in chronic and acute neuropathologies. Neural Plasticity, 1-21.
  • Chávez-Castillo, M., Rojas, M., & Bautista, J. (2017). Excitotoxicity: an organized crime at the cellular level. Archivos de Medicina, 8(3), 198-203.
  • Choi, N. Y., Choi, H., Park, H. H., Lee, E. H., Yu, H. J., Lee, K. Y., & Koh, S. H. (2014). Neuroprotective effects of amlodipine besylate and benidipine hydrochloride on oxidative stress-injured neural stem cells. Brain Research, 1551, 1-12.
  • Dong, X. X., Wang, Y., & Qin, Z. H. (2009). Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacologica Sinica, 30(4), 379-387.
  • Erel, O. (2004). A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clinical Biochemistry, 37(4), 277-285.
  • Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103-1111.
  • Giansante, G., Marte, A., Romei, A., Prestigio, C., Onofri, F., Benfenati, F., & Valente, P. (2020). Presynaptic L-type Ca2+ channels increase glutamate release probability and excitatory strength in the hippocampus during chronic neuroinflammation. Journal of Neuroscience, 40(36), 6825-6841.
  • Godfraind, T. (2017). Discovery and development of calcium channel blockers. Frontiers in Pharmacology, 8, 286-301.
  • Godoy, J. A., Rios, J. A., Picón-Pagès, P., Herrera-Fernández, V., Swaby, B., Crepin, G., & Muñoz, F. J. (2021). Mitostasis, Calcium and Free Radicals in Health, Aging and Neurodegeneration. Biomolecules, 11(7), 1012-1022.
  • Higley, M. J., & Sabatini, B. L. (2012). Calcium signaling in dendritic spines. Cold Spring Harbor Perspectives in Biology, 4(4), a005686.
  • Hu, Y., Li, J., Liu, P., Chen, X., Guo, D. H., Li, Q. S., & Rahman, K. (2012). Protection of SH-SY5Y neuronal cells from glutamate-induced apoptosis by 3, 6′-disinapoyl sucrose, a bioactive compound isolated from Radix Polygala. Journal of Biomedicine and Biotechnology, 38, 1-5.
  • Khurana, K., & Bansal, N. (2019). Lacidipine attenuates reserpine-induced depression-like behavior and oxido-nitrosative stress in mice. Naunyn-Schmiedeberg's Archives of Pharmacology, 392(10), 1265-1275.
  • Khurana, K., Kumar, M., & Bansal, N. (2021). Lacidipine Prevents Scopolamine-Induced Memory Impairment by Reducing Brain Oxido-nitrosative Stress in Mice. Neurotoxicity Research, 39, 1-16.
  • Kim, T. Y., Niimi, K., & Takahashi, E. (2016). Role of Cav2. 1 Channel Signaling in Glutamate-Related Brain Injury. Brain Disorder Therapy, 5(226), 2-22.
  • Kim, Y. H., Eom, J. W., & Koh, J. Y. (2020). Mechanism of zinc excitotoxicity: a focus on AMPK. Frontiers in Neuroscience, 14, 958-966.
  • Kimura, M., Katayama, K., & Nishizawa, Y. (1999). Role of glutamate receptors and voltage-dependent calcium channels in glutamate toxicity in energy-compromised cortical neurons. The Japanese Journal of Pharmacology, 80(4), 351-358.
  • Krasil’Nikova, I., Surin, A., Sorokina, E., Fisenko, A., Boyarkin, D., Balyasin, M., & Pinelis, V. (2019). Insulin protects cortical neurons against glutamate excitotoxicity. Frontiers in Neuroscience, 13, 1027-1035.
  • Kritis, A. A., Stamoula, E. G., Paniskaki, K. A., & Vavilis, T. D. (2015). Researching glutamate–induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Frontiers in Cellular Neuroscience, 9, 91-103.
  • Lee, Y. M., He, W., & Liou, Y. C. (2021). The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide. Cell Death & Disease, 12(1), 1-13.
  • Mahajan, A. S., Babbar, R., Kansal, N., Agarwal, S. K., & Ray, P. C. (2007). Antihypertensive and antioxidant action of amlodipine and vitamin C in patients of essential hypertension. Journal of Clinical Biochemistry and Nutrition, 40(2), 141-147.
  • Manev, H., Kharlamov, E., Uz, T., Mason, R. P., & Cagnoli, C. M. (1997). Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells. Experimental Neurology, 146(1), 171-178.
  • Sanchez, A. B., Medders, K. E., Maung, R., Sánchez-Pavón, P., Ojeda-Juárez, D., & Kaul, M. (2016). CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l-type Ca 2+ channels upstream of p38 MAPK. Journal of Neuroinflammation, 13(1), 1-12.
  • Stuiver, B. T., Douma, B. R. K., Bakker, R., Nyakas, C., & Luiten, P. G. M. (1996). In vivo protection against NMDA-induced neurodegeneration by MK-801 and nimodipine: combined therapy and temporal course of protection. Neurodegeneration, 5(2), 153-159.
  • Sun, Z. W., Zhang, L., Zhu, S. J., Chen, W. C., & Mei, B. (2010). Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage. Neuroscience Bulletin, 26(1), 8-16.
  • Taghizadehghalehjoughi, A., & Naldan, M. E. (2018). The study of diazepam, pregabalin and glucose effect on glutamate toxicity: In vitro study. Medicine, 7(4), 797-801.
  • Vallazza-Deschamps, G., Fuchs, C., Cia, D., Tessier, L. H., Sahel, J. A., Dreyfus, H., & Picaud, S. (2005). Diltiazem-induced neuroprotection in glutamate excitotoxicity and ischemic insult of retinal neurons. Documenta Ophthalmologica, 110(1), 25-35.
  • Valtcheva, S., & Venance, L. (2019). Control of long-term plasticity by glutamate transporters. Frontiers in Synaptic Neuroscience, 11, 10-22.
  • Wheeler, D. B., Randall, A., & Tsien, R. W. (1996). Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca2+ channels in rat hippocampus. Journal of Neuroscience, 16(7), 2226-2237.
  • Yang, X. R., Sun, P., Qin, H. P., Si, P. P., Sun, X. F., & Zhang, C. (2012). Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons. Acta physiologica Sinica, 64(6), 609-616.
  • Zyśk, M., Gapys, B., Ronowska, A., Gul-Hinc, S., Erlandsson, A., Iwanicki, A.,& Bielarczyk, H. (2018). Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells. PLoS One, 13(12), e0209363.
There are 32 citations in total.

Details

Primary Language English
Journal Section Articles
Authors

Betül Çiçek 0000-0003-1395-1326

Ali Taghizadehghalehjoughi 0000-0002-3506-0324

Ahmet Hacımüftüoğlu 0000-0002-9658-3313

Aysegul Yılmaz 0000-0001-5843-1661

Publication Date March 31, 2022
Submission Date October 3, 2021
Acceptance Date February 23, 2022
Published in Issue Year 2022 Volume: 7 Issue: 1

Cite

APA Çiçek, B., Taghizadehghalehjoughi, A., Hacımüftüoğlu, A., Yılmaz, A. (2022). The Difference Between The Response To Glutamate Excitotoxicity and The Role Of Ca2+ Channel Blockers in Cortical Neuron and SH-SY5Y Cells Cultures. Journal of Anatolian Environmental and Animal Sciences, 7(1), 45-52. https://doi.org/10.35229/jaes.1003400


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