Name: measurement of cytosolic ca2+ in isolated contractile lymphatics
Uploaded: 2011-12-08T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

<p class="jove_content"&gt; Эта работа выполнена при поддержке гранта NIH P20RR018766 и грант ABMRF / Фонд Алкоголь исследований.</p

1. Животные <ol><li> Все процедуры были утверждены по Уходу за животными и использованию Комитетом на Университет штата Луизиана Центра медицинских наук и были проведены в соответствии с руководящими принципами Национального института здоровья (NIH публикация № 85-12, пересмотренная 1996). Му...

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Новую комбинацию методов был применен для изучения внутренней перекачки лимфатических сосудов. Возможность одновременно измерять изменения [Са 2 +] я и диаметром в насосных лимфатических позволит исследований относительный вклад Ca 2 +-зависимый и Са 2 +-информирован...

<table border="1"><tbody><tr><td> 1. Рингер 5x со </td></tr><tr><td Химический </td><td> Компания </td><td> Номер в каталоге </td><td> Количество </td></tr><tr><td> Хлористый натрий </td><td> EMD </td><td> SX0420-3 </td><td> 35 г </td></tr><tr><td> Хлористый калий </td><td> JT Baker </td><td> 3040 </td><td> 1,75 г </td></tr><tr><td> Хлорид кальция </td><td> Сигма </td><td> C-3881 </td><td> 1,47 г </td></tr><tr><td> Сульфат магния </td><td> Сигма </td><td> М-9397 </td><td> 1,44 г </td></tr><tr><td> Стерильные фильтрованной воды </td><td> N / A </td><td> N / A </td><td> Довести до 1 л </td></tr><tr><td> Стерильные фильтр в автоклаве бутылки и сохраняет при температуре 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. MOPS буфера </td><td></td><td></td><td></td></tr><tr><td> Химический </td><td> Компания </TD&gt; <td> Номер в каталоге </td><td> Количество </td></tr><tr><td> MOPS </td><td> Сигма </td><td> M3183 </td><td> 125,6 г </td></tr><tr><td> Стерильные фильтрованной воды </td><td> N / A </td><td> N / A </td><td> Довести до 1 л </td></tr><tr><td> Стерильные фильтр в автоклаве бутылки и сохраняет при температуре 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. Альбумин физиологическим раствором соли (АПСС) </td></tr><tr><td> Химический </td><td> Компания </td><td> Номер в каталоге </td><td> Количество </td></tr><tr><td> Рингер акций (5x) </td><td> N / A </td><td> N / A </td><td> 200 мл </td></tr><tr><td> Швабры буфера </td><td> N / A </td><td> N / A </td><td> 5 мл </td></tr><tr><td> Фосфат натрия </td><td> Сигма </td><td> S-9638 </td><td> 0,168 г </td></tr><tr><td> Натрий Пируват </td><td> Сигма </td><td> P5280 </td><td> 0,22 г </td></tr><tr><td> ЭДТА натрия салт </td><td> Сигма </td><td> ED2SS </td><td> 0,0074 г </td></tr><tr><td> Глюкоза </td><td> Сигма </td><td> G7528 </td><td> 0,901 г </td></tr><tr><td> Альбумин, говядину </td><td> USB </td><td> 10856 </td><td> 10 г </td></tr><tr><td> Стерильные фильтрованной воды </td><td> N / A </td><td> N / A </td><td> Довести до 1 л </td></tr><tr><td> Отрегулируйте рН до 7,4 при 37 ° С, то стерильный фильтр в автоклаве бутылки и хранить при 4 ° C. </td></tr></tbody></table> Таблица 1. Конкретные используемые реагенты. Храните все при 4 ° C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Введем подхода к оценке цитозольного Са<sup&gt; 2 +</sup&gt; Концентрация в отдельных лимфатических изучать Са<sup&gt; 2 +</sup&gt;-Зависимый и Са<sup&gt; 2 +</sup&gt;-Информирование механизмы лимфатической гладких мышц.

Измерение цитозольного Са 2 + В изолированном Сократительная лимфатических

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

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Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

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