İskemi ve reoksijenizasyonun neden olduğu protein S100B salıverilmesi: Glutamatın koruyucu etkisinin mekanizması
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Date
2013-06-07
Authors
Demircan, Celaleddin
Journal Title
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Publisher
Uludağ Üniversitesi
Abstract
Amaç: S100B, özellikle astrositlerde sentezlenen ve salıverilen kalsiyum bağlayıcı bir proteindir. Salıverilme mekanizması iyi bilinmemekle beraber, iskeminin S100B salıverilmesini artırdığı gösterilmiştir. Daha önce in vitro beyin dilimleri modeli kullanarak yaptığımız çalışmalarda, iskemi ve reoksijenizasyonun (REO) neden olduğu S100B salıverilmesindeki artışın glutamat tarafından önlendiğini göstermiştik. Çalışma glutamatın bu etkisinin mekanizmasını aydınlatmak amacıyla planlanmıştır. Yöntem: Sıçan beyninden hazırlanan kortikal dilimler preinkubasyon dönemi sonrası 60 dk iskemik ortamda (oksijen ve glukoz yok), ardından oksijen ve glukoz içeren ortamda (REO) inkübe edildi.Daha sonra toplanan inkubasyon ortamları dilimlerden salıverilen S100B ve laktat dehidrogenaz (LDH) ölçümü için kullanıldı. Glutamatın S100B ve LDH salıverilmesi üzerine etkileri a-ketoglutarat, laktat, piruvat ve 2-ketobutirat gibi maddelerin etkileri ile karşılaştırılarak incelendi. Bulgular: İskemi ve REO, S100B salıverilmesini sırasıyla %81 ve %244 oranında, REO ise LDH çıkışını %192 oranında artırdı. Ortama glutamat eklenmesi, S100B ve LDH artışlarını anlamlı şekilde önledi. Enerji substratı ve serbest oksijen radikal (SOR) tutucu etkileri olan a-ketoglutarat, piruvat ve laktat ile sadece enerji substratı olan 2-ketobutirat, glutamat benzeri etki gösterdiler. Dilimlerin hücrede SOR oluşturan menadion ile inkübe edilmeleri, S100B ve LDH salıverilmesinde önemli artışa neden oldu. Glutamat ve çalışmada kullanılan diğer maddeler menadion nedenli bu artışı önlediler Sonuç: Çalışmanın sonuçları, a-ketoglutarat ve diğer a-ketoasitlerin iskemi, REO ve menadion nedenli S100B ve LDH artışları üzerine glutamat benzeri etkiye sahip olduklarını göstermektedir. a-Ketoglutarat, glutamatın bir metabolik ürünü olduğu için glutamatın koruyucu etkisinin, onun enerji substratı olması yanısıra SOR tutucu etkisine de bağlı olduğunu düşündürmektedir.
Objective: S100B is a calcium-binding protein especially expressed in and released from astrocytes. Although release mechanism is not clear, it has been shown that ischemia increases S100B release. In our earlier studies performed in vitro brain slices, we showed that ischemia and subsequent reoxygenation (REO)-induced increases on S100B release were prevented by glutamate. This study was planned to elucidate the mechanism of decreasing effect of glutamate on S100B release. Methods: After preincubation period, cortical slices prepared from rat brain were incubated 60 min in ischemic medium (oxygen-glucose deprivation, OGD) and then in the medium containing oxygen and glucose (REO). At the end of this periods, incubation media were collected and used for the measurement of S100B and lactate dehydrogenase (LDH) released from the slices. The effects of glutamate on S100B and LDH release were tested by comparing with the effects of other substances such as ?-ketoglutarate, lactate, pyruvate and 2-ketobutirate. Results: Ischemia and REO increased release of S100B by 81% and 244% respectively, and REO increased LDH leakage by 192%. The addition of glutamate into the medium significantly prevented increments on S100B and LDH outputs. a-Ketoglutarate, pyruvate and lactate that have energy substrate and free oxygen radical scavenger effects and 2-ketobutirat those are energy substrates and also having free oxygen radical scavenger activities and 2-ketobutirate that is only an energy substrate, showed glutamate-like effects. When slices were incubated with menadion that generates free oxygen radicals intracellularly caused significant increases on S100B and LDH outputs. Glutamate and other substances tested in the study protected the slices against menadion-induced these increments. Conclusion: The results presented here demonstrate that a-ketoglutarate and other a-ketoacids exerted a similar effect as glutamate on OGD, REO and menadion-induced S100B and LDH outputs. Since a-ketoglutarate is a metabolic product of glutamate, the protective effect of glutamate is probably due to its free radical scavenger properties in addition to its energy substrate properties.
Objective: S100B is a calcium-binding protein especially expressed in and released from astrocytes. Although release mechanism is not clear, it has been shown that ischemia increases S100B release. In our earlier studies performed in vitro brain slices, we showed that ischemia and subsequent reoxygenation (REO)-induced increases on S100B release were prevented by glutamate. This study was planned to elucidate the mechanism of decreasing effect of glutamate on S100B release. Methods: After preincubation period, cortical slices prepared from rat brain were incubated 60 min in ischemic medium (oxygen-glucose deprivation, OGD) and then in the medium containing oxygen and glucose (REO). At the end of this periods, incubation media were collected and used for the measurement of S100B and lactate dehydrogenase (LDH) released from the slices. The effects of glutamate on S100B and LDH release were tested by comparing with the effects of other substances such as ?-ketoglutarate, lactate, pyruvate and 2-ketobutirate. Results: Ischemia and REO increased release of S100B by 81% and 244% respectively, and REO increased LDH leakage by 192%. The addition of glutamate into the medium significantly prevented increments on S100B and LDH outputs. a-Ketoglutarate, pyruvate and lactate that have energy substrate and free oxygen radical scavenger effects and 2-ketobutirat those are energy substrates and also having free oxygen radical scavenger activities and 2-ketobutirate that is only an energy substrate, showed glutamate-like effects. When slices were incubated with menadion that generates free oxygen radicals intracellularly caused significant increases on S100B and LDH outputs. Glutamate and other substances tested in the study protected the slices against menadion-induced these increments. Conclusion: The results presented here demonstrate that a-ketoglutarate and other a-ketoacids exerted a similar effect as glutamate on OGD, REO and menadion-induced S100B and LDH outputs. Since a-ketoglutarate is a metabolic product of glutamate, the protective effect of glutamate is probably due to its free radical scavenger properties in addition to its energy substrate properties.
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Keywords
İskemi, Reoksijenasyon, S100B proteini, Laktat dehidrogenaz, Glutamat, α-ketoglutarat, Ischemia, Reoxygenation, S100B protein, Lactate dehydrogenase, Glutamate, α-ketoglutarate
Citation
Demircan, C. (2013). İskemi ve reoksijenizasyonun neden olduğu protein S100B salıverilmesi: Glutamatın koruyucu etkisinin mekanizması. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.