Summary

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Published: December 08, 2011
doi:

Summary

我们介绍的方法来评价细胞质CA<sup> 2 +</sup>孤立淋巴集中在研究CA<sup> 2 +</sup>依赖和Ca<sup> 2 +</sup>敏淋巴管平滑肌收缩机制。

Abstract

淋巴管组成一个多功能的交通运输系统保持流体的动态平衡,血脂中央流通,作为一个潜在的有害抗原的监控系统,优化粘膜免疫和适应性免疫反应1。从组织间液进入盲端的初始淋巴管,然后是对运输压力梯度较大的收集淋巴管形成淋巴。每个收集淋巴是由一系列称为lymphangions段,二尖瓣阀门,防止回流隔开。每个lymphangion拥有一个收缩的周期,推动反压力梯度向中央循环 2淋巴结。这种阶段性的收缩模式类似心动周期,收缩压和舒张压阶段,并具有较低的收缩频率4。此外,淋巴管平滑肌产生提示音并显示生肌的收缩和扩张,我ñ回应管腔压力的增加和减少,分别为5。因此,建议同时支持淋巴管的阶段性和补品收缩的分子机制的混合体。

平滑肌收缩一般是受细胞质的Ca 2 +浓度(内[Ca2 +] i的),加上灵敏度的Ca 2 +,在周围的细胞6环境变化的响应收缩元素。的[Ca 2 +] 是运动的Ca 2 +通过质膜配体或电压门控离子通道的释放和吸收的Ca 2 +从内部商店相结合确定。胞质的Ca 2 +结合到钙调蛋白和酶的激活,如肌球蛋白轻链(MLC)的激酶(MLCK),这反过来又磷酸化MLC的肌动蛋白-肌球蛋白介导的收缩 8 。然而,这种通路的敏感性钙<sup> 2 +可调节的MLC磷酸酶(MLCP)9。 MLCP活性受Rho激酶(ROCK)和肌球蛋白磷酸酶抑制蛋白CPI – 17。

在这里,我们目前超过隔离,淋巴管灌注时间的方法,以评估变化的[Ca 2 +] 为了研究的Ca 2 +依赖和Ca 2 +敏淋巴管平滑肌收缩机制。采用离体大鼠肠系膜淋巴管收集,我们研究的[Ca 2 +]拉伸引起变化我和收缩活动。孤立淋巴模型提供了优势的压力,流量,和化学成分的沐浴液,可以严格控制。的[Ca 2 +] 是通过加载比例的Ca 2 +结合染料FURA – 2淋巴确定。这些研究将提供新的方法来学习不同的分子机制,规范阶段性的更广泛的问题进补收缩与淋巴管平滑肌的收缩。

Protocol

1。动物机构动物护理和使用委员会在路易斯安那州立大学健康科学中心的所有程序批准,并按照国家卫生研究所(NIH出版物,编号85-12,1996年修订)的指引执行。雄性SD大鼠(查尔斯河实验室,体重270-350克)被安置在一个控制温度(22℃)和控制的照明(12:12 h光照明暗周期)环境。抵达后,将大鼠提交了为期一周的驯化期间,提供了标准的大鼠议员(2018 Teklad全球18%的蛋白质鼠类饮食,…

Discussion

一个新颖的方法组合研究内在的淋巴管抽水。能够同时测量变化的[Ca 2 +] i和抽水淋巴管的直径将允许 Ca 2 +依赖 Ca 2 +敏的信号通路在整体管收缩淋巴循环的机制。相对贡献的研究

淋巴管收缩周期由过音叠加的阶段性收缩。数据表明,每一个阶段性的收缩与中的[Ca 2 +] i的瞬态上升,这表明一个核心作用?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

<p class="jove_content"这项工作是由美国国立卫生研究院授予P20RR018766和酒精研究从ABMRF /基金会授予的支持。</p

Materials

<tdChemical
1. Ringer 5x Stock
Company Catalog Number Amount
Sodium Chloride EMD SX0420-3 35 g
Potassium Chloride J.T. Baker 3040 1.75 g
Calcium Chloride Sigma C-3881 1.47 g
Magnesium Sulfate Sigma M-9397 1.44 g
Sterile Filtered Water N/A N/A Bring to 1 L
Sterile filter into autoclaved bottles and stores at 4 °C
       
2. MOPS buffer      
Chemical Company Catalog Number Amount
MOPS Sigma M3183 125.6 g
Sterile Filtered Water N/A N/A Bring to 1 L
Sterile filter into autoclaved bottles and stores at 4 °C
       
3. Albumin Physiological Salt Solution (APSS)
Chemical Company Catalog Number Amount
Ringer stock (5x) N/A N/A 200 mL
Mops Buffer N/A N/A 5 mL
Sodium Phosphate Sigma S-9638 0.168 g
Sodium Pyruvate Sigma P5280 0.22 g
EDTA sodium salt Sigma ED2SS 0.0074 g
Glucose Sigma G7528 0.901 g
Albumin, Bovine USB 10856 10 g
Sterile Filtered Water N/A N/A Bring to 1 L
Adjust pH to 7.4 at 37° C, then sterile filter into autoclaved bottles and store at 4 °C.

Table 1. Specific Reagents Used. Store all at 4 °C.

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Citazione di questo articolo
Souza-Smith, F. M., Kurtz, K. M., Breslin, J. W. Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics. J. Vis. Exp. (58), e3438, doi:10.3791/3438 (2011).

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