Acute ethanol exposure prevents PMA-mediated augmentation of N-methyl-D-aspartate receptor function in primary cultured cerebellar granule cells.
Acute ethanol exposure prevents PMA-mediated augmentation of N-methyl-D-aspartate receptor function in primary cultured cerebellar granule cells.
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急性乙醇暴露可防止原代培养的小脑颗粒细胞中 PMA 介导的 N-甲基-D-天冬氨酸受体功能增强。
DOI：
10.1016/j.alcohol.2011.03.002
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Popp,RLisa
中科院分区：
文献类型：
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作者：
Reneau,Jason;Reyland,MaryE;Popp,RLisa
许多细胞内蛋白质和信号级联有助于天然N-甲基-d-天冬氨酸受体（NMDAR）的乙醇敏感性。一种推定的蛋白质是丝氨酸/苏氨酸激酶，蛋白激酶C（PKC）。本研究的目的是评估PKC是否调节原代培养的小脑颗粒细胞（CGC）中表达的天然NMDAR的乙醇敏感性。采用全细胞膜片钳技术，我们评估了乙醇对NMDA诱导电流（INMDA）的抑制（100μM NMDA +10μM甘氨酸）在CGC中是否被改变，其中新的和经典的PKC亚型被佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）激活。10、50或100 mM乙醇对NMDA诱导的稳态电流幅度（ISS）或在CGC中表达的NMDAR的峰值电流幅度（IPk）的抑制百分比，其中PKC在37°C下通过12.5分钟、100 nM PMA暴露激活，与从对照细胞中包含的受体获得的电流没有差异。然而，PMA介导的增强IPkin乙醇的情况下被取消后，短暂的应用程序的10或1 mM乙醇coapplied激动剂，并观察到这种抑制增强受体功能后乙醇暴露长达8分钟。因为我们先前已经证明PMA介导的NMDAR的INMDA增加是通过激活PKCα来实现的，所以我们评估了乙醇（1、10、50和100 mM）对PKCα活性的影响。1 mM乙醇使PKCα活性降低18%，并且活性随着乙醇浓度的增加而降低，在100 mM乙醇中观察到50%的抑制。这些数据表明，乙醇破坏PMA介导的INMDA增加可能是由于乙醇降低PKCα活性。然而，考虑到PKCα活性的不完全阻断和观察到这种现象的低浓度乙醇，其他乙醇敏感性信号级联也必须参与。
Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine/threonine kinase, protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100μM NMDA plus 10μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50, or 100mM ethanol of NMDA-induced steady-state current amplitudes (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5min, 100nM PMA exposure at 37°C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPkin the absence of ethanol was abolished after brief applications of 10 or 1mM ethanol coapplied with agonists, and this suppression of enhanced receptor function was observed for up to 8min post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDAof NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50, and 100mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDAmay be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved.