Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Road, Auckland, New Zealand.
Neurotoxicology. 2010 Mar;31(2):204-14. doi: 10.1016/j.neuro.2010.01.001. Epub 2010 Jan 12.
TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca(2+) permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1-CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H(2)O(2) (600 microM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 microM) or buthionine sulfoximine (BSO; 4 microM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 microM; MitoE 2 microM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd(3+) 500 microM; Ruthenium red 1 microM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 microM), N-(p-amylcinnomoyl)anthranilic acid (ACA, 25 microM) or flufenamic acid (FFA, 200 microM) failed to protect pyramidal neurons from damage caused by exogenous H(2)O(2), and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers.
TRPM2 和 TPPV4 通道是 TRP 通道家族的两个成员，它们被广泛认为在大脑中表达，但它们的确切表达模式和功能尚不清楚。由于它们的高钙离子通透性和由活性氧物质（TRPM2）、细胞肿胀、低 pH 值和高温（TRPV4）门控，它们可能与各种脑病理学相关的细胞损伤有关。本研究的目的是研究这些通道的表达及其在器官型海马切片培养物中氧化应激诱导的细胞损伤中的潜在作用，该模型保留了神经元和星形胶质细胞之间的复杂相互作用。通过 RT-PCR 和 Western blot 确认通道表达，免疫细胞化学显示 TRPM2 在 CA1-CA3 锥体神经元中，TRPV4 在星形胶质细胞中。外源性应用 H2O2（600μM）诱导的氧化应激导致锥体神经元的选择性损伤，而用 mercaptosuccinate（MCS；400μM）或 buthionine sulfoximine（BSO；4μM）诱导的氧化应激主要损伤星形胶质细胞，如碘化丙啶荧光所示。抗氧化剂（Trolox 500μM；MitoE 2μM）减少了神经元和星形胶质细胞的死亡。TRPV4 通道阻断剂（Gd3+ 500μM；ruthenium red 1μM）增加了 MCS 或 BSO 处理后星形胶质细胞的活力，与这些通道的表达模式一致。TRPM2 通道阻断剂克霉唑（20μM）、N-（对氨基肉桂酰基）anthranilic 酸（ACA，25μM）或 flufenamic 酸（FFA，200μM）未能保护锥体神经元免受外源性 H2O2 引起的损伤，并增加了 MCS 和 BSO 引起的这些神经元的损伤。在对不同形式的氧化应激具有明显不同易感性的海马神经元和星形胶质细胞中，应激敏感的 TRPM2 和 TRPV4 通道的差异表达表明这些通道特异性参与氧化应激诱导的细胞损伤。然而，由于缺乏 TRPM2 通道阻断剂的保护作用，TRPM2 通道激活与细胞死亡之间的确切关系仍有待确定。
