【作者】 郑海锋；

【导师】 许文燮； 李在琉；

【作者基本信息】 延边大学 ， 生理学， 2004， 硕士

【摘要】 作为细胞膜的重要组成成分，不饱和脂肪酸不仅对保持生物膜的流动性与稳定性具有很重要的作用，而且还可以调节某些细胞信号转导，从而影响细胞功能。不饱和脂肪酸包括多不饱和脂肪酸和单不饱和脂肪酸，花生四烯酸（arachidonic aicd, AA），亚油酸（linoleic acid, LA）属于ω-6多不饱和脂肪酸，油酸（oleic acid, OA）属于ω-9单不饱和脂肪酸。这些不饱和脂肪酸对离子通道的调节非常复杂，它们既可以通过改变通道蛋白构象或与通道蛋白周围的细胞膜作用，影响离子通道的功能；又可以通过其环氧化酶、脂氧化酶和单加氧酶的代谢产物途径、蛋白激酶途径、以及其他细胞信号转导途径影响离子通道的功能。对于不同离子通道，不饱和脂肪酸的作用途径是不同的。本研究室前期研究发现，花生四烯酸等不饱和脂肪酸对豚鼠胃窦平滑肌细胞氯电流，L型钙电流以及毒蕈碱电流均有直接抑制作用。但是，不饱和脂肪酸对钙激活钾电流(calcium-activated potassium current, I_K（Ca）)的影响及其作用机制尚不清楚。因此，本文的研究目的在于：在用2型胶原酶急性分离的豚鼠胃窦环行肌上，利用膜片钳技术的全细胞记录法观察外源性不饱和脂肪酸对豚鼠胃窦平滑肌细胞钙激活钾电流的影响，并探讨其作用机制。 本文的主要实验结果如下： 1．膜电位钳制在60.0mV，电极内液加人0.1 gmol L^-1GTA，以每10s的时间间隔给予20mV的阶跃刺激使细胞膜电位从-40mV去极化到+100mV，持续时间440ms，记录出外向性钙激活钾电流。花生四烯酸（arachidonic acid, AA）明显增强I_K（Ca），并具有量效关系。当AA的浓度在2、5和10μmol L^-1，膜电位去极化至+60mV时，分别增强，I_K（Ca）15.9±3.6％、31.9±7.0％和46.3±10.4％。 2．为了进一步确定AA增强的是钙激活钾电流部分，记录了自发瞬间外向电延边大学医学院生理学硕士学位论文摘要 流（Spontaneous transient outward eurrents，sme、）—主要是由钙激活钾 通道开放引起。使用与前面相同的电极内液，膜电位钳制在一20mV，便可 记录到smes。10林molL，AA明显增强smes。3.在同样实验条件下，另一种不饱和脂肪酸，亚油酸（li noleic acid，LA）也 增强几（ca），而且也有明显量效关系。5、10和20卿ofL一，LA，在膜电位 去极化至+6 omV时，分别增强场ca)27.8士4名%、37.9士13.9%和70.8士 19.9%。4.为探讨脂肪酸细ca)增强效应与不饱和脂肪酸的不饱和度之间的关系，在本 实验中观察了10“molL，的从、LA和油酸（oleicac记，oA）对细ca) 的影响。结果发现，在膜电位去极化至+6 omv时，它们分别增强细ca) 46.3 士10.4%、37.9士13.9%和13.5士5.1%，即AA（C20:4，eis一5，8，11，14） >LA（C 18:2，eis一9，12）>OA（C18:l，eis一9）。5.为了探讨不饱和脂肪酸增强细ca)的作用是通过直接途径还是其它间接途径 实现，做了以下实验，结果发现: （1）H一7（蛋白激酶C抑制剂）10林molL一‘预处理以后，给予阶跃性去极 化刺激使膜电位去极化至+6 omv时，10林molL一‘从仍能增强细ca) 41 .8士3.7%。与对照值相比较无显著性差异（P>0 .05）。 （2）用indome仇acin‘环氧化酶抑制剂)10林molL‘和17一octadecynoie acid（细胞色素P450抑制剂）预处理后，给予阶跃性去极化刺激使膜电位去 极化至+6 OmV时，同一浓度的AA仍增强缅ca)分别为42.9士10.8%和40.8 幼.8%。与对照值相比较无显著性差异护>0.05)。 （3）用nordihydroguaiaretie acid（脂氧化酶抑制剂）一。林molL一‘预处理 后，给予阶跃性去极化刺激使膜电位去极化至+6 omv时，同一浓度AA增 强细ca)11 .3料.3%，其作用明显被抑制，与对照值相比较有显著性差异 （P<0 .05）。 上述结果提示:1.不饱和脂肪酸明显增强钙激活钾电流，并且有量效关系;2.不饱和脂肪酸中双键数目越多，增强效应越强;3.不饱和脂肪酸脂氧化酶代谢途径参与调节不饱和脂肪酸增强钙激活钾电流的过程。更多还原

【Abstract】 Being an important componet of plasma membrane, unsaturated fatty acids can not only keep plasma membrane fluidity and stability, but also affect cell functions through modulating some signal transduction. Unsaturated fatty acids include ployunsaturated fatty aicds and monounsaturated fatty acids. Arachidonic acid (AA) and linoleic acid (LA) are to -6 polyunsaturated fatty acids, while oleic acid (OA) is to -9 monounsaturated acid. The modulation of these unsaturated fatty aicds on ion channels is complicated. They can affect ion channels via both the interaction between fatty acids and ion channels protein or the interference with plasma membrane and their cycooxygenase, lipoxygenase, epoxygenase metabolites or cellular signal transduction pathways. In our previous study, we have reported that AA and other unsaturated fatty acids directly inhibited calcium current (ICa), Cl- current (ICl) and muscarinic current (ICCh). However, the effect of unsaturated on calcium-activated potassium current and the mechanism are not clear.In this study, the effect of unsaturated fatty acids on calcium-activated potassium current and its mechanism were investigated in gastric antral circular myocytes of guinea pig. The results are as below:1. Under the whole cell configuration, the membrane potential was clamped at -60 mV. When pipette solution contained egtazic acid 0.1 mmol L-1, IK(Ca) was elicited by step voltage command pulse from -40 mV to +100mV for 440ms with a 20mV increment at 10 sec intervals. An unsaturated fatty acid, arachidonic acid (AA, with 4 double bonds) significantly increased IK(Ca) in a dose-dependant manner. AA increased IK(Ca) by 15.9 3.6%, 31.9 + 7.0%, 46.3 + 10.4% at 2, 5, and 10 mol L-1 at +60 mV, respectively.2. Under the whole patch clamp mode and with the same pipette solution, the holding potential was clamped at -20 mV. The spontaneous transient outward currents (STOCs) was then recorded. 10 mol L-1 AA markedly increased STOCs.3. Another unsaturated fatty acid, linoleic acid (LA, with 2 double bonds) also increased IK(ca) by 27.8 4.8%, 37.9 13.9% , 70.8 + 19.9% at the concentration of 5, 10, and 20 mol L-1 at +60 mV, respectively.4. To determine the inhibitory potency of unsaturated fatty acids, the effects of different unsaturated fatty acids on IK(Ca) were compared. Under the whole-cell configuration, the same concertration (10 mol L"1) of AA, LA, and oleic acid (OA, one double bond) increased /K(ca) by 46.3 10.4%, 37.9+ 13.9% and 13.5 + 5.1% at +60mV, respectively . The increased potency sequence was AA (C20: 4, cis-5, 8, 11, U)>LA (CIS: 2, cis-9, ]2)>OA (C18: 1, cis-9).5. In the presence of H-7 (protein phosphorylation C inhibitor, 10 fimol L"1) 10 Hmol/L AA still increased /K(Ca> by 41.8 3.7 % at +60mV. There was no significant difference between two groups before and after pretreatment with H-7. H-7 also not blocked AA-induced increase of STOCs. In the presence of indomethacin (Indo, the cyclooxygenase inhibitor, 10 nmol L"1) and 17-octadecynoic acid (17-ODA, cytochrome P450 inhibitor, 10 |imol L"1), 10 \imol L"1 AA increased /K(ca) by 42.9 10.8 % and 40.8 6.8 % at +60mV.There was no significant difference between two groups before and after pretreatment with Indo or 17-ODA. However, in the presence of nordihydroguaiaretic acid(NDGA, the lipoxygenase inhibitor, 10 mol L"1), 10 mol L’1 AA increased /K(Ca) by 1 1.3 4.3% at +60mV. There was no significant difference between two groups before and after pretreatment with NDGA.The conclusion: 1) /K(Ca) was increased by unsaturated fatty acid in dose-dependant manner; 2) There was a correlation between the degree of cis unsaturation and the increasing potency of /K(Ca) by unsaturated fatty acids; 3) Unsaturated fatty acid-induced increase of /K(ca) was mediated by lipoxygenase metabolism pathway更多还原