Lipski Janusz, Park Thomas I H, Li Dong, Lee Stanley C W, Trevarton Alexander J, Chung Kenny K H, Freestone Peter S, Bai Ji-Zhong
Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, 92-019 Auckland, New Zealand.
Brain Res. 2006 Mar 10;1077(1):187-99. doi: 10.1016/j.brainres.2006.01.016. Epub 2006 Feb 17.
During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.
在体内急性缺血期间或体外氧 - 葡萄糖剥夺（OGD）时，CA1神经元去极化、肿胀并钙超载。我们的目的是检验这一假设：对OGD的初始反应至少部分归因于瞬时受体电位（TRP）通道的激活。由于一些TRP通道对温度敏感，我们还比较了通道的药理学阻断作用与降低温度的作用。从Wistar大鼠获取的急性海马脑片（350μm）被置于36℃的人工脑脊液（ACSF）中。使用细胞外、细胞内或全细胞膜片钳记录对CA1神经元进行电生理监测。通过记录相对组织电阻的变化评估细胞肿胀，并在用fura - 2葡聚糖加载神经元后测量细胞内钙的变化。TRP通道阻滞剂（钌红、La3 +、Gd3 +、2 - 氨基乙氧基二苯硼酸酯（2 - APB））或温度降低3℃可减少对OGD的反应。这包括：（a）细胞外直流电位负向偏移的延迟增加；（b）初始缓慢膜去极化速率降低、OGD诱导的细胞输入电阻增加的发展减慢以及全细胞内向电流的发展减慢；（c）组织肿胀减轻；（d）细胞内钙升高幅度减小。当测量细胞外直流偏移的延迟时，轻度低温（33℃）与La3 +或Gd3 +（100μM）显示出阻断效应。通过逆转录 - 聚合酶链反应（RT - PCR）在CA1亚区证实了TRPM2/TRPM7（对氧化应激敏感）和TRPV3/TRPV4（对温度敏感）通道的表达。这些结果表明，TRP或TRP样通道被细胞应激激活，并导致缺血诱导的膜去极化、细胞内钙积累和细胞肿胀。我们还假设，通过降低温度关闭一些TRP通道（TRPV3和/或TRPV4）可能部分解释了低温的神经保护作用。
