Hiperglisemi RBL-1 hücrelerinde Ca2+ salınımı ile aktive olan Ca2+ akımlarını (ICRAC) arttırır

Amaç: Depo bağımlı Ca2+ girişi (SOCE) olarak farklı türde akımlar tanımlanmış olsa da bunlar arasında en iyi bilineni Ca2+ salınımı ile aktive olan Ca2+ akımlarıdır (ICRAC). SOCE yolağı çeşitli biyolojik süreçleri düzenleyen hücre içi Ca2+ ([Ca2+]i) sinyalleri oluşturmak için önemli bir yoldur. Anormal SOCE, özellikle alerji, diyabet, yangı ve kanseri içeren birçok çeşitli hastalık tipinin altında yatan önemli sebeplerden biri olarak görülmektedir. Bu çalışmada, hipergliseminin SOCE aktivitesi üzerine olan etkileri elektrofizyolojik yöntemlerle incelenmiştir. Yöntem: Bu çalışmada, sıçan bazofilik lösemi-1 (RBL- 1) hücreleri yüksek glikoz (YG, 50mM) ve normal glikoz (5.5mM) derişimine sahip DMEM solüsyonları kullanılarak çoğaltılmıştır. RBL-1 hücreleri yeterli sayıya ulaşmasının ardından 72 saat süreyle yüksek glikoz ortamına maruz bırakılmıştır. Bu süreyi takiben hücrelerden Fura-2 tabanlı Ca2+ görüntüleme tekniği ile SOCE aktivitesi ve voltaj kenetleme yöntemiyle ICRAC akımları ölçülmüştür. Bulgular: ICRAC akım ölçümleri patch-clmap ve Fura-2 tabanlı Ca2+ görüntüleme yöntemleriyle gerçekleştirilmiştir. Pozitif kontrol deneyler için PYR6 (SOCE blokörü) kullanılarak akımların SOCE yolağı üzerinden olduğu doğrulanmıştır. Ayrıca hipergliseminin RBL-1 hücrelerinde SOCE aktivitesini kontrol değerlerine göre istatistiksel olarak anlamlı derecede arttırdığı iki farklı yöntemle tespit edilmiştir. Sonuç: Bulgularımız diyabetik patolojilerde karşılaşılan Ca2+ homeostazındaki ve ER/SR depolarının Ca2+ içeriğindeki bozulmalarda SOCE yolağı ile ilgili proteinlerin önemli bir rolü olabileceğini ortaya koymaktadır. Hücre düzeyinde elde edilen bu bulgular sınırlı olsa da ileride geliştirilecek olan çalışmalara büyük katkılar sağlayacağı düşünülmektedir.

Hyperglycemia increases Ca2+ realese activated Ca2+ current (ICRAC) in RBL-1 cells

Objective: Although different types of currents have been defined as store-operated Ca2+ entry (SOCE), the best characterized of these is Ca2+ release-activated Ca2+ currents (ICRAC). The SOCE pathway is an important way to generate cytosolic Ca2+ signals that regulate various biological processes. Abnormal SOCE appears to be one of the underlying causes of several diseases, particularly allergies, diabetes, inflammation, and cancer. In this study, the effects of hyperglycemia on SOCE activity were investigated by electrophysiological methods. Methods: In this study, rat basophilic leukemia-1 (RBL-1) cells were grown using DMEM solutions with high glucose (HG, 50mM) and normal glucose (5.5mM) concentrations. Following 72 h incubation period, SOCE activity was measured by Fura-2 based Ca2+ imaging technique and ICRAC currents recorded by voltage clamp method. Results: ICRAC current measurements were performed by the patch-clmap method and Fura-2 based Ca2+ imaging methods. For positive control experiments, using the PYR6 (SOCE blocker), it was confirmed that the currents were through the SOCE pathway. In addition, it was determined by two different methods that hyperglycemia significantly increased SOCE activity in RBL-1 cells compared to control values. Conclusion: Our findings reveal that the SOCE pathway may have an important role in the disturbances in Ca2+ homeostasis and Ca2+ content of ER/SR stores encountered in diabetic pathologies. Although these findings at the cellular level are limited, it is thought that they will make great contributions to future studies.

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