这项工作是由国家心脏，肺和血液研究所（NIH R01-HL089564）的资助KLB和博士后奖学金由美国心脏协会（09PRE2260209）和Arthur J.施密特基金会BKM。

1。手术切除的小肠肠系膜血管商场<ol><li>麻醉300-400克SD大鼠（4％）与异氟醚吸入给药。 </li><li>执行中线剖腹手术暴露的小肠系膜。 Exteriorize通过腹部切口非常小心，以避免创伤暴露的肠管及肠系膜小肠和大肠。轻轻地扇了的肠系膜超过无菌纱布。 </li><li>外科手术切除小肠和大肠的一部分，包括盲肠（沿利润率和肠系膜上动脉/静脉）从主分支的起源仔细剪裁血管。 </li><li>切除的组织转移到烧杯中含有冰冷解剖溶液（ 表1）。 </li><li>切除的小肠，大鼠安乐死，而在麻醉状态下。 </li></ol> 2。解剖一个ð清洁动脉<ol><li>转移切除小肠和肠系膜街机冷却的解剖室内（4-5°C）含有夹层的解决方案。该室包括一个100毫米的玻璃陪替氏培养皿用SYLGARD 184弹性体基容纳解剖标签;维持在4-5℃的循环水浴冷却的碱是被放置在陪替氏培养皿。 </li><li>使用的整个肠道手术剪刀剪下约10-12厘米的段。处切断的肠段保持其脉管连接的每一端，然后移除未使用的从腔室离开一个段及其连接的血管（肠系膜街机）孤立肠。 </li><li>牵制的小肠段，在近前侧的腔室，使得主分支肠系膜上动脉/静脉是在从中心（ 图1）的中心，并在随后的订单的分支辐射扩频肠系膜。 PL<html>王牌引脚上的肠段（而不是血管）。 </li><li>区分其相对明亮的红色，厚厚的墙壁和特性的V形分支，而不是U形见于静脉的分支动脉静脉。 </li><li>可视化通过解剖显微镜的照明，仔细清除脂肪组织和结缔组织的周围的第二 -四阶动脉接近（在图1中的虚线圆），使用微型钳和剪刀肠子。控股邻近的静脉，有利于清除脂肪组织和结缔组织。避免不必要的动脉延伸。 </li><li>肠系膜动脉血管的结缔组织清除就可以被分离出，并用于细胞分离（单细胞膜片钳技术研究，第3-4）或插管的压力myography（第5章）。剪切分支点之间的线段，通常最长为1厘米的长度。 </li></ol>jove_title"&gt; 3。细胞分离膜片钳研究<ol><li>准备0.5 L的隔离解决方案（ 见表1），并把它分成2份，然后再调整pH值。的第一部分（通常为100毫升）应被加热至37℃下，pH值应调节到7.2，在37℃下（37°C隔离解决方案）。的第二部分的分离解决方案应被冷却到4℃，pH值应调整至7.2，在4℃（冰镇的分离解决方案）。在pH调节后，这两种解决方案的渗透压必须被调整，通过添加适当量的D-葡萄糖或H 2 O至298毫渗</li><li>转让2毫升37°C隔离解决方案，以小玻璃瓶中，在37°C孵育步骤3.4使用。加入20毫克的牛血清白蛋白（BSA），10毫升37°C隔离解决方案，解散，重新加热到37°C，并调整pH值至7.2。 2毫升上述溶液中添加4毫克型胶原酶VIII（Sigma公司，批号089K8612:633ü尼特/毫克胶原酶活性，285单位/ mg Caseinase的活动，1.6单位/ mg梭菌活动），0.64毫克弹性蛋白酶IV型（Sigma公司，批号110M7025V 5.9单位/毫克，加入40μl的16毫克/毫升的股票，准备在37°。 C隔离解决方案）1和2μl中号DL-二硫苏糖醇（DTT Sigma公司）预暖的酶液37°C，在准备步骤3.5。 </li><li> 2-3段的肠系膜动脉转移到含2毫升冰冷的隔离解决方案和地方的冰盘上的35毫米组织培养皿中。切断肠系膜上动脉段为3-4毫米长的碎片。 </li><li>使用巴斯德吸管与火抛光头（防止动脉血管的损害）转移到一个玻璃小瓶（15×45毫米，1 DRAM），含2毫升预热的37°C的动脉段隔离解决方案，并培育30分钟，37 °C。 </li><li> 30分钟后，小心吸37°C的隔离解决方案使用巴斯德吸管，加2毫升温水酶溶液，室温放置35分钟在37℃下</li><li>酶溶液中孵育35分钟后，立即在冰面上，将小瓶吸酶溶液和添加2毫升冰冷的隔离解决方案（重复的愿望和新鲜的冰镇隔离解决方案增加了4倍）。然后，在一个体积为1毫升的冰镇隔离解决方案， 轻轻捣碎动脉段与抛光的巴斯德吸移管释放出单个的肠系膜动脉平滑肌的细胞（MASMCs）的10-20倍。 </li><li>定期验证心肌细胞外观上的35毫米的菜，将一滴细胞悬液，在显微镜下观看。健康的MASMCs应该有一个平滑细长的外观。不是所有的在动脉壁的细胞将被释放。将细胞悬浮液应保存在冰中。 </li><li> MASMC健康的另一项有用的指标是生理的Ca 2 +浓度的耐受性。浴小号填写录音室（生物科学工具，CSC-25L）辨率含有2mM CaCl 2的 （组合物在室温（RT）的pH值为7.3， 见表1）和位置琼脂桥（用2％琼脂的2 M氯化钾弯曲玻璃毛细管填充，参比电极插入（华纳仪器，REF -3L））箱内。使用抛光的巴斯德吸移管，约100-200微升细胞悬浮液转移到一个记录室，使细胞粘附15分钟。完好无损的细胞不应该在2mM的显着收缩的CaCl 2的溶液（的细胞应该出现是细长的或棒状成球，而不是向上舍入）。 </li><li>第一等分的细胞悬浮液后加入到记录室，浴溶液中的0.25毫升含有2mM CaCl 2的应该被添加到细胞悬浮液的剩余量。在此解决方案中，这种其它的细胞悬浮液可保持在冰上，并用于膜片钳记录长达6小时。 </li></ol> 4。使用全细胞膜片钳测量的KV7钾电流或膜电位<ol><li>后心肌细胞坚持的记录室（25毫米编号1轮盖玻片，华纳仪器）的玻璃基体，启动的浴溶液（ 表1）的连续灌注。记录的KV7电流的KV1和KV2家庭的延迟整流钾电流的隔离，沐浴液，应补充与100μMGDCL。 </li><li>制作从:硼硅酸盐玻璃（Kimax-51，金布尔大通）使用吸移管牵拉和microforge的一个补丁移液器;MΩ内部溶液（ 表1）充满时其电阻应是4-5。外套的下1/3的吸移管（〜1-2毫米以上的前端），SYLGARD 184。 </li><li>对于记录的KV7电流MASMCs，应采用穿孔膜片钳全​​细胞电压钳技术。使用的穿孔修补程序配置有助于防止depletio的n的膜磷脂酰肌醇4,5 -二磷酸（PIP 2）和随后的快（在5分钟内）破旧的KV7的电流，也就是通常观察到使用常规的破裂补丁配置。补编内部溶液（ 表1）与120微克/毫升两性霉素B:3μl的2毫克/毫升两性霉素B库存（在DMSO中制备并保持冻结在小等分试样和避光）添加到0.5毫升的内部溶液。填充的补丁移液管的前端与两性霉素B无内部溶液通过移液管尖中的两性霉素B无内部溶液浸渍，然后在另一端进行抽吸，使用10毫升注射器附着通过一个管件（的Tygonŕ 3603）。加入两性霉素B含吸管的解决方案使用的Eppendorf Microloader移液器吸头的顶部，直到移液器，约有一半的充满溶​​液。删除任何气泡，轻轻拍打移液管。立即用移液管。 </li><li>将滴管到所述电极保持器，并使用显微操作降低到一个细长的心肌细胞的表面上（接近，但不是在细胞核中， 图2的顶部）的吸移管。当移液管阻力增加6〜10MΩ，适用吸力，实现了千兆密封（&gt; 10GΩ）。 </li><li>设置保持电压为0毫伏，而等待膜穿孔。应用100毫秒的电压的步骤至+10 mV，然后到-10 mV，使用放大器来补偿快速移液器电容瞬变。的外观，全细胞电容的瞬变，小持续正电流（通常为2-10 PA）的同时，将指示存在的功能KV7渠道。 </li><li>成功与两性霉素B穿孔会降低接电阻为35MΩ或更少。当前记录，然后可以开始使用5秒-4 mV的保持电压的电压阶跃协议。我们使用-4 mV的保持电压，以实现接近最大延迟同属的其他类型的失活tifier钾电流（主要由KV1和KV2通道介导的），否则将污染的记录的相对小的非灭活KV7电流。长（5秒）的电压阶跃协议是要实现稳态激活KV7的渠道，这很慢，电压依赖性激活和失活。 KV7电流的持续电压步骤期间不灭活。 </li><li>要记录KV7电流 - 电压（IV）关系，适用于5秒的电压阶跃到+16 mV的电压范围从-84毫伏，在哪些的KV7通道的开放概率最小，在哪些KV7通道的开放概率达到高原，在每个测试电压，5秒后退一步，以保持电压（-4毫伏），持续10秒。这将需要约350分钟的全系列的测试脉冲（ 图3A）。执行2-3控制IV录音，以确保记录的电流随时间是稳定的。情节平均端的脉冲电流（平均电流记录的最后1秒）对电压创建的IV曲线（ 图3D）。如果电流是稳定的，控制的IV曲线应约叠加。 </li><li>在应用任何药物开始连续记录电流在-20 mV的一个时间过程设计的协议。至少5分钟的药物应用之前，确保稳定的记录的电流。应用药物为5-15分钟，或直至达到一个稳定状态（ 图3C），然后终止该时间过程协议和记录两个IV曲线，使用的电压步协议。冲蚀的药物，并记录药物清洗过程中的时间，然后通过记录两个额外的IV曲线。 </li><li>在实验结束后，10的μMlinopirdine或10μM的XE991，不可逆抑制剂KV7通道的应用，这些药物应充分抑制记录在负-20毫伏（ 图3D）的电压的电流。 </li><li&gt;每个实验后用洗涤剂，乙醇和热水清洗室和灌注系统，以除去所用的药物的任何痕迹。 </li></ol> 5。使用压力Myography完整的动脉段<ol><li>购自丹麦的，MYO技术（DMT A / S，丹麦，型号110P）的压力肌动描记系统用于的压力myography实验。安装在不锈钢腔动脉段插入到一个端部和可动到另一端的合适的玻璃套管的水平固定的左玻璃套管。两个压力换能器（权的P1和P2上的左侧）是建立在以监测动脉管腔内的压力。将被用于正确的玻璃套管的流体从动脉内的压力调整到其近似的生理水平。首先，预填充开放10ml注射器（用作一个重力进料压力柱），与管腔溶液（ 表1）。提高注射器推解决方案TION，通过聚乙烯管材照顾到右边的玻璃套管，油管或套管，以避免空气中的气泡。左边的套管应预先填写流明解决方案，通过一个1毫升注射器。填充压力肌动描记室容器浴溶液（ 表1）用10ml。松散暂停前的每一侧的玻璃套管上的细的尼龙缝合线。 </li><li>小心转移解剖肠系膜动脉段（通常为3 次或4 阶段，约200 - 350微米的直径;从步骤2.6）从解剖腔肌动描记的压力室，通过保持到该段的一端部上，使用微镊子。 </li><li>可视化通过照明解剖显微镜，按住动脉段的一端，并轻轻将它滑到的左玻璃套管和使用的尼龙缝合线固定结束。轻轻冲洗管腔液通过安装的容器，在容器内，以清除血液和碎片。左边的注射器的阀，然后应关闭，使得这一边提出反对从右侧被调节的压力流体静态列。 </li><li>使用显微操作器，位置更接近可移动的合适的玻璃套管的动脉段和拉段在它的右端，最后固定​​它与尼龙缝合线。剪掉多余的缝合线。 </li><li>在舞台上安装的压力肌动描记单位的压力肌动描记显微镜。将盖在压力肌动描记室。盖的腔室中包含的的灌流入口和连接到它的吸入管。连接灌流进气歧管，使灌流室温浴缸或60毫米KCl溶液通过重力的压力肌动描记室。试验物质，如被发​​现在膜片钳测量影响KV7通道功能的药物或激素，一般是直接施加浴溶液中，不经由浴支持erfusion或在管腔溶液。将使用沐浴液灌流，洗出的KCl溶液。的吸入管连接到真空系统，以除去过量的灌流。所述温度传感器插入到浴溶液中，通过该开口设置在肌动描记器的腔室盖。 </li><li>连接的压力肌动描记单元的肌醇接口系统，硬件的myoview软件（DMT），使通信的肌动描记单元。 </li><li>打开的myoview的软件在电脑上。在"参数"窗口中，设置目标温度为35°C的温度公差为1°C，80毫米汞柱和80毫米汞柱的目标流出压力的目标流入压力。加载校准文件和校准的容器外径，使动脉直径的变化，准确地记录在微米。调整对比度必要的安装部分的映衬下，使一个很好的观点。如何使用软件的详细说明设置在用户手册从DMT。 </li><li>逐步提高用10ml注射器（作为一个重力进料压力柱），时间为15分钟以上一个，直到P1压力传感器读数为80毫米汞柱。虽然提高的压力列，检查是否有泄漏的容器。如果有任何泄漏，丢弃段，装入另一个动脉段（重复步骤从5.2）。调整套管必要时之间的距离。应调整的距离，在这样一种方式，加压动脉段大约是直的（但是未拉伸），并形成了一个肩类似的关系（ 图4A）的图案。 </li><li>开启压力肌动描记室的加热通过在的肌接口系统的控制。允许浴溶液逐渐升温，直到它达到36-37℃。 </li><li>的使用的XYZ调整的目标中的DMT显微镜必要时保持容器在焦点和方便准确地跟踪的ch法兰外血管直径。 </li><li>短暂灌流（约30秒），60 mM KCl中，检查安装的容器的可行性。一个可行的血管收缩加入KCl和迅速扩张后，立即冲洗60毫米氯化钾（superfuse约60毫升的沐浴液洗出的氯化钾）。在本试验中，调整的浴溶液，以确切地是10毫升的体积。浴的温度降低的KCl的测试和冲洗过程中，因为这些解决方案是在室温下，但腔室的加热器将还原温度为36-37℃下，在几分钟之内。 </li><li>允许动脉平衡至少30分钟（直径应该是稳定的，至少在最后10分钟在此期间）。添加浓缩的试验物质，以直接的浴溶液，轻轻的来回移液混合浴溶液中的试验物质，得到适当的最终浓度在10毫升的腔室容积的腔室的边缘附近。陈另外的测试物质（s）中的血管直径以下的水电站可以监视在实况视频图像，并在图像分析窗口，同时也绘制实验正在进行中。 </li><li>实验完成后，所存储的数据文件（*。肌）可以打开作为一个电子表格文件，作进一步的分析。 </li></ol> 6。有代表性的结果 KV7电流从肠系膜动脉肌细胞，使用前一个电压阶跃协议（ 图3A，B黑色痕迹）和在治疗过程中与KV7通道活化剂锌吡啶硫酮（ZnPyr，100nM的， 图3A，B所示的结果在图3中示出典型的录音红色痕迹）。 3细胞相似的电压的步骤的数据进行平均，以制备的电流-电压（IV）曲线， 如图3D所示。一位代表过程中提高电流幅值测量连续的电压钳制在-20 mV的白枕克治疗与100nM的锌吡啶硫酮在图3C中所示， 图4B中示出的时间过程的效果的ZnPyr上肠系膜动脉的外径使用压力myography测量容器100 PM精 ​​氨酸加压素（AVP）是预缩之前除了100纳米ZnPyr。在图4C中示出的平均剂量-反应数据ZnPyr诱导肠系膜动脉扩张。 <img src="/files/ftp_upload/4263/4263fig1.jpg" alt="图1"/> 图1。的肠系膜商场，在解剖室内的画面。 <img src="/files/ftp_upload/4263/4263fig2.jpg" alt="图2"/> 图照片的心肌细胞在其表面上与补丁移液器。 <img src="/files/ftp_upload/4263/4263fig3.jpg" alt="图3"/> 图3。原始的痕迹KV7电流，时间疗程的药<html>应用程序，IV曲线。 A.家庭响应于电压的步骤（前面板）治疗前（控制，黑色）和以下稳定在100nM的ZnPyr（红色）的存在下，在一个单一的MASMC中记录在连续电流的痕迹（下面板）。 B.代表电流的痕迹A（用箭头表示），控制（黑色）和100:NM ZnPyr（红色）。灰色条表示的时间间隔平均电流幅值测量电流 - 电压（IV）曲线。在右边的箭头表示跨步电压。 C. KV7 100nM的ZnPyr电流增强的时间过程中测量具有连续的电压钳位在-20毫伏（巴）。 D.平均IV曲线组（n = 3）来自KV7电流密度之前记录的（对照组，实心圆），在治疗过程中与100nM的ZnPyr（空心圆），并用10μMXE991（实心三角形）的治疗过程中。减去非特异性泄漏电流帕斯莫尔等人所描述的:1。 ENT"&gt; <img src="/files/ftp_upload/4263/4263fig4.jpg" alt="图4"/> 图4。:ZnPyr，诱导放宽预缩与100 PM AVP的肠系膜动脉。 A.安装在照片的肠系膜上动脉的压力肌动描记的设置。 B.该船只的外径变动的时间过程中响应于应用100 PM AVP（开放式酒吧）随后应用的100nM的ZnPyr的（填充巴）。 C.条形图总结的100纳米ZnPyr引起的vasodilaion的结果。

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没有利益冲突的声明。

这里所描述的方法和实验方法相当强劲，并且能够产生明确的和可重复的结果时一丝不苟的细节。良好的电生理记录和收缩/扩张动脉段是依赖于细胞和动脉血管的健康。细胞制剂可以改变每一天，甚至使用相同的协议。隔离解决方案，可用于长达2周，但如果细胞制备的质量低，随之而来的两个天应准备新的隔离解决方案。我们已经发现，一批一批，因此隔离条件（时间的培养和酶的浓度）酶活性的变化需要进行优化与每一批新的酶。我们还发现，此处介绍的用于大鼠肠系膜动脉的细胞分离协议将不是最佳的其他血管床或其他物种，可以具有不同的混合或窝点性的胞外基质成分。每个血管的准备都需要有自己的优化的情况产生健康的细胞。 ，我们发现最有帮助的确定健康细胞的标准是:1）细胞形态:外观流畅，细长的，但略有收缩后捣碎MA段的隔离解决方案，其中包含50μM的Ca 2 +，并保持相同的外观，当转移到2 mM的Ca 2 +的包含外部溶液。并非所有的细胞将保留其几乎完全放松的细长形状，但应被用于电生理记录只有近充分放松肌细胞; 2）细胞应是光学致密的，这表示一个完整的未损坏的膜; 3）用于健康肌细胞的静息膜电压记录钾电流或膜电压电流钳记录应该是更负比-40 mV时（通常在-45 mV的范围内根据离子条件德-56毫伏cribed在我们这里的协议）。 为了确保成功的电生理记录，沐浴液和液（成分见表1）应准备在当天的实验。这是至关重要的调节重量克分子渗透压浓度的内部和浴的解决方案是相同的（298-299毫渗）。 等压小动脉（&lt;500微米）使用压力myography，更接近生理条件比等长收缩力测量动脉功能测量用钢丝肌动描记。肌动描记在一个压力的船舶通常是更敏感的血管收缩激动剂2-4，更接近5星，6在体内测量的敏感性。低浓度的激动剂的敏感性增加，能够查明的有关生理皮摩尔浓度的AVP诱导的信号转导机制7，8。 压力myography保留容器的几何形状，和维护的血管内皮细胞的完整性。内皮细胞介导的作用的药物可区别于平滑肌-介导的效应，在完整和内皮剥蚀船只9通过进行并行测量。可以通过物理损坏的内皮细胞（ 例如，通过一个人的头发来回通过管腔）或灌注动脉的管腔内的空气通过血管内皮细胞的故意中断。血管内皮功能的丧失（而不是血管平滑肌功能），应确认测量减少内皮细胞介导的血管扩张反应，对乙酰胆碱的血管收缩激动剂9。 并行肠系膜上动脉肌细胞离子电流/膜电压的测量采用膜片钳技术和动脉收缩/肠系膜arteri扩张ES使用压力myography可以使特定的离子通道在生理信号转导级联以及治疗药物的行动中阐明的角色。治疗针对特定类别的离子通道或特定的信号转导的中间体可以应用，相加或序列提供这些信道在细胞功能的调节，在细胞水平上，在完整的动脉的收缩/扩张的作用机制有关下列内容的信息。治疗方法，增强或抑制动脉直径与相应的效果上的特定类型的离子电流的收敛结果，提供了更可靠的解释，可以得到比使用上述两种方法本身。理想情况下，所研究的代理的浓度依赖性，应确定在两个系统中。为了评估相关生理或治疗，检测的浓度应与浓度的生理环境oŕ实现临床治疗。作为这些类型的实验方法，可以发现在我们以前的出版物8-10。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td>评论</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S5886 </td><td>剖析解决方案:145 沐浴液电:140 电:10内部解决方案 为心肌细胞的隔离解决方案:140 浴的压力myography解决方案:145 流明的压力myography解决方案:145 </td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P5405 </td><td>剖析解决方案:4.7 沐浴液电:5.36 电:135内部解决方案 隔离解决方案肌:5.36 浴的压力myography解决方案:4.7 流明的压力myography解决方案:4.7 </td></tr><tr><td>钾EGTA </td><td>西格玛</td><td> E4378 </td><td>电:0.05内部解决方案</td></tr><tr><td> HEPES </td><td>西格玛</td><td> H9136 </td><td>沐浴液电:10 电:10内部解决方案 隔离解决方案，为心肌细胞*:10 </td></tr><tr><td>磷酸氢二钠</td><td>西格玛</td><td> S5136 </td><td>隔离解决方案，为心肌细胞*:0.34 </td></tr><tr><td>磷酸氢二钾</td><td>西格玛</td><td> P5655 </td><td>隔离解决方案，为心肌细胞*:0.44 </td></tr><tr><td>氯化镁</td><td>西格玛</td><td> M2393 </td><td>沐浴液电*:1.2 内部电:1解决方案 隔离解决方案肌:1.2 </td></tr><tr><td>氯化钙</td><td>西格玛</td><tD&gt; C7902 </td><td>沐浴液电:2 为心肌细胞的隔离解决方案:0.05 </td></tr><tr><td>磷酸钠</td><td> Fisher Scientific则</td><td> BP331-1 </td><td>剖析解决方案:1.2 浴的压力myography解决方案:1.2 流明的压力myography解决方案:1.2 </td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M2643 </td><td>剖析解决方案:1.17 浴的压力myography解决方案:1.17 流明的压力myography解决方案:1.17 </td></tr><tr><td> MOPS </td><td> Fisher Scientific则</td><td> BP308 </td><td>剖析解决方案: 浴的压力myography解决方案:3 流明的压力myography解决方案:3 </td></tr><tr><td>丙酮酸</td><td>西格玛</td><td> P4562 </td><td>剖析解决方案: 沐浴解决方案压力myography:2 流明的压力myography解决方案:2 </td></tr><tr><td> EDTA二水</td><td>研究有机物</td><td> 9572E </td><td>剖析解决方案:0.02 浴的压力myography解决方案:0.02 流明的压力myography解决方案:0.02 </td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G7021 </td><td>剖析解决方案: 沐浴液电:10 电:20内部解决方案 隔离解决方案，为心肌细胞*:10 浴的压力myography解决方案:5 流明的压力myography解决方案:5 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912 </td><td>剖析解决方案:1％ 流明压力myography溶液:1％ </td></tr><tr><td> pH值</td><td></td><td></td><td>剖析搜索解决方案N:7.4 沐浴液电*:7.3 电:7.2的内部解决方案 隔离解决方案肌:7.2 浴的压力myography解决方案:7.4 流明的压力myography解决方案:7.4 </td></tr><tr><td>渗透压</td><td></td><td></td><td>剖析解决方案:300 沐浴液电:298 电:298内部解决方案 隔离解决方案肌:298 浴的压力myography解决方案:300 流明的压力myography解决方案:300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> 表1中，在实验中使用的解决方案的组件。 </td></tr></tbody></table>

exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography

电阻动脉的壁内的平滑肌细胞的收缩或松弛确定动脉直径并由此控制的血液流至容器内，并有助于全身血压。收缩过程中被限制主要是由胞内游离钙浓度（〔Ca 2 +] cyt的），它是依次由多种离子转运和频道控制。离子通道是常见的中间体，在信号转导通路激活血管活性激素对血管收缩或舒张功能。离子通道往往是有针对性的治疗药物，无论是有意（ 如钙离子通道阻断剂诱导的血管舒张和降低血压）或无意的（ 如引起不必要的心血管副作用）。 KV7（KCNQ）电压激活的钾离子通道最近被牵连的生理和治疗的重要塔尔格ETS调节平滑肌收缩。为了阐明KV7渠道在生理信号转导和治疗药物的行动中的具体作用，我们需要研究他们的活动是如何在细胞水平上调节，以及评估他们的贡献的背景下，完整的动脉。 大鼠肠系膜动脉提供了一个有用的模型系统。动脉可以很容易地解剖，结缔组织清除，并用孤立的动脉肌细胞膜片钳电生理准备，或为血管收缩剂/血管舒张反应的在相对生理条件下测量插管，加压。在这里，我们描述了这两种类型的测量方法，并提供一些例子，实验一体化设计可提供更清楚地了解这些离子通道在调节血管张力的作用。

Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

测量KV7（KCNQ）钾离子通道活性的孤立动脉细胞（中与测量大蟒/扩张的反应（使用压力myography），同时采用膜片钳电生理技术）可以揭示有关的重要信息的的KV7渠道作用在血管平滑肌生理和药理学。

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

exploring-arterial-smooth-muscle-kv7-potassium-channel-function-using

Nanyang Technological University

Research

JoVE Journal

Biology

15.1K 次观看.  Loyola University Chicago. 测量KV7（KCNQ）钾离子通道活性的孤立动脉细胞（中与测量大蟒/扩张的反应（使用压力myography），同时采用膜片钳电生理技术）可以揭示有关的重要信息的的KV7渠道作用在血管平滑肌生理和药理学。

视频: Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

科研

医学

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

生物学

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

微电极镀膜法记录从可吸入性中脑动脉的膜电位

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

通过全细胞膜片钳技术 记录 H9c2心肌细胞上的电压依赖性钾电流

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

摘要

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此视频中的章节

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

分离，培养和成年小鼠Cardiomyoctyes的功能分析

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

微电极镀膜法记录从可吸入性中脑动脉的膜电位

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

通过全细胞膜片钳技术记录 H9c2心肌细胞上的电压依赖性钾电流

体外细胞生理学研究中视网膜微动脉的分离

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

摘要

探索更多视频

此视频中的章节

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

分离，培养和成年小鼠Cardiomyoctyes的功能分析

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

微电极镀膜法记录从可吸入性中脑动脉的膜电位

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

通过全细胞膜片钳技术 记录 H9c2心肌细胞上的电压依赖性钾电流

体外细胞生理学研究中视网膜微动脉的分离

制备和利用新鲜分离的人类分离器平滑肌细胞，用于9-苯酚敏感阳离子电流的表征

通过全细胞膜片钳技术记录 H9c2心肌细胞上的电压依赖性钾电流