Kintner Douglas B, Look Andy, Shull Gary E, Sun Dandan
Department of Neurosurgery, University of Wisconsin Medical School, H4/332 Clinical Sciences Center, 600 Highland Ave., Madison, Wisconsin 53792, USA.
Am J Physiol Cell Physiol. 2005 Oct;289(4):C934-45. doi: 10.1152/ajpcell.00092.2005. Epub 2005 May 18.
We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na+ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by approximately 75% in NHE1+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 microM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na+ but also Ca2+ homeostasis via reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX led to a 103% increase in intracellular Ca2+ concentration ([Ca2+]i) in NHE1+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca2+]i by 73%. To further investigate changes of Ca2+ signaling, bradykinin-mediated Ca2+ release was evaluated. Bradykinin-mediated intracellular Ca2+ transient in NHE1+/+ astrocytes was increased by approximately 84% after OGD/REOX. However, in NHE1-/- astrocytes or NHE1+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca2+ release was increased by only approximately 34%. Inhibition of the reverse mode of Na+/Ca2+ exchange abolished OGD/REOX-mediated Ca2+ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na+ accumulation subsequently alters Ca2+ homeostasis via Na+/Ca2+ exchange.
我们最近报道，在氧糖剥夺（OGD）后，星形胶质细胞中的钠氢交换体1（NHE1）活性受到刺激并导致细胞内钠负荷增加。然而，这种NHE1活性刺激的潜在机制及其对星形胶质细胞功能的影响尚不清楚。在本研究中，我们研究了ERK1/2信号通路在NHE1激活中的作用。在OGD 2小时和复氧（REOX）1小时后，NHE1+/+星形胶质细胞中的NHE1活性升高了约75%。30 microM的PD-98059部分阻断了OGD/REOX介导的NHE1刺激。在OGD/REOX后，在NHE1+/+星形胶质细胞中检测到磷酸化ERK1/2的表达增加。此外，NHE1活性的刺激不仅破坏了钠稳态，还通过钠钙交换的反向模式破坏了钙稳态。在毒胡萝卜素存在的情况下，OGD/REOX导致NHE1+/+星形胶质细胞内钙浓度（[Ca2+]i）增加103%。用NHE1抑制剂HOE-642抑制NHE1活性可使OGD/REOX诱导的[Ca2+]i升高降低73%。为了进一步研究钙信号的变化，评估了缓激肽介导的钙释放。在OGD/REOX后，NHE1+/+星形胶质细胞中缓激肽介导的细胞内钙瞬变增加了约84%。然而，在NHE1-/-星形胶质细胞或用HOE-642处理的NHE1+/+星形胶质细胞中，缓激肽诱导的钙释放仅增加了约34%。抑制钠钙交换的反向模式消除了OGD/REOX介导的钙升高。总之，我们的数据表明，ERK1/2参与了体外缺血后星形胶质细胞中NHE1的激活。NHE1介导的钠积累随后通过钠钙交换改变钙稳态。
