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首页> 外文期刊>Journal of Neuroscience Methods >Electroporation loading and photoactivation of caged InsP(3): tools to investigate the relation between cellular ATP release in response to intracellular InsP(3) elevation.
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Electroporation loading and photoactivation of caged InsP(3): tools to investigate the relation between cellular ATP release in response to intracellular InsP(3) elevation.

机译：关在笼中的InsP（3）的电穿孔加载和光激活：研究细胞内InsP（3）升高反应中细胞ATP释放之间关系的工具。

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摘要

Photolytic liberation of InsP(3) in single cells triggers cell-to-cell propagating calcium changes that are communicated by a gap junctional and a paracrine purinergic pathway involving InsP(3)-triggered ATP release. We investigated the relation between the InsP(3) stimulus and the resulting ATP release in ECV304 cells using UV photolysis of caged compounds and bioluminescent ATP measurements. Careful consideration of all steps, starting from caged InsP(3) loading into the cells by electroporation, up to photoliberation upon UV exposure, allowed to derive a dose-response relation that revealed a first part with a flattening ATP release response in the below 10microM InsP(3) concentration range and a second phase of steeply increasing ATP release in response to above 10microM InsP(3) stimulation. ATP release triggered by below 10microM InsP(3) concentrations attained a level in the order of 30% above baseline ATP release, while the steeply increasing response to high InsP(3) concentrations attained values in the order of 150% above baseline. Our data indicate the involvement of low affinity InsP(3) receptor sites in the pathway leading to triggered ATP release, with activation of these receptors causing the release of 1-2% of the total cellular ATP pool.
机译：InsP（3）在单细胞中的光解解放触发细胞间传播的钙变化，该变化通过间隙连接和涉及InsP（3）触发的ATP释放的旁分泌嘌呤能途径传递。我们调查了InsP（3）刺激和由此产生的ATP释放在ECV304细胞中的关系，使用的是笼养化合物的紫外光解和生物发光ATP测量。仔细考虑所有步骤，从通过电穿孔将笼状InsP（3）加载到细胞中，直到紫外线暴露后光解，可以得出剂量反应关系，揭示了第一部分具有低于10microM的平坦ATP释放响应InsP（3）浓度范围和急剧增加的ATP释放的第二阶段，以响应以上10microM InsP（3）刺激。低于10microM InsP（3）浓度触发的ATP释放水平达到比基线ATP释放高30％的水平，而对高InsP（3）浓度的响应急剧增加，达到高于基线约150％的水平。我们的数据表明，低亲和力的InsP（3）受体位点参与了触发ATP释放的途径，这些受体的激活导致了总细胞ATP池的1-2％的释放。

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