Effects of frequently applied carbon monoxide releasing molecules (CORMs) in typical CO-sensitive model systems - A comparative in vitro study.
Effects of frequently applied carbon monoxide releasing molecules (CORMs) in typical CO-sensitive model systems - A comparative in vitro study.
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DOI：
10.1016/j.abb.2020.108383
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发表时间：
2020-04
影响因子：
3.9
通讯作者：
David Stucki;Heide Krahl;Moritz Walter;Julia Steinhausen;K. Hommel;P. Brenneisen;W. Stahl
中科院分区：
文献类型：
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作者：
David Stucki;Heide Krahl;Moritz Walter;Julia Steinhausen;K. Hommel;P. Brenneisen;W. Stahl
细胞内一氧化碳（CO）是一种气体信号分子，由血红素加氧酶在血红素降解为胆绿素时酶促产生。细胞内产生的CO的靶结构是血红素蛋白，包括呼吸链的细胞色素氧化酶、细胞色素P450依赖性单加氧酶或肌红蛋白。对于CO信号传导的研究，可获得不同结构的CO释放分子（CORM）。在这里，三个常用的CORM（CORM-2，CORM-3和CORM-401）的性质，以提供CO在生物测试系统和地址敏感血红素蛋白进行了研究。在肌红蛋白结合测定中研究了CO释放，发现CORM-2-和CORM-3的CO释放是快速的（<5分钟），而CORM-401连续提供CO（>50分钟）。还用肌红蛋白测定法评估了CORM储备溶液的储存稳定性。只有CORM-401储备溶液在7天内是稳定的。CORM与重组细胞色素P450孵育导致酶活性的抑制。然而，只有CORM-3和CORM-401被证明适用于该测试系统，因为使用失活的CORM-2（iCORM-2）的对照也导致酶活性丧失。采用高分辨率呼吸仪和细胞外通量技术研究了CORM对呼吸链的影响。在第一种方法中，CORM-2和CORM-3与氧测量发生干扰，因为即使没有细胞存在时，也在培养基中检测到氧的快速耗尽。然而，CORM-401不干扰氧测量，并且观察到预期的细胞呼吸抑制。CORM-2不适合用于细胞外通量技术的氧测量，CORM-3的应用在该系统中没有显示出任何效果。然而，CORM-401观察到细胞呼吸的CO依赖性抑制。基于本实验得出的结论是，CORM-401产生最可靠的CO特定的结果调制典型的CO目标。CORM-2和CORM-3不适合研究CO对细胞内血红素基团的生物学效应。根据实验设置，应谨慎评估这些化合物获得的数据。
Intracellular carbon monoxide (CO) is a gaseous signaling molecule and is generated enzymatically by heme oxygenases upon degradation of heme to billiverdin. Target structures for intracellular produced CO are heme proteins including cytochromecoxidase of the respiratory chain, cytochrome P450-dependent monooxygenases, or myoglobin. For studies on CO signaling, CO-releasing molecules (CORMs) of different structure are available. Here, three frequently used CORMs (CORM-2, CORM-3 and CORM-401) were studied for their properties to provide CO in biological test systems and address susceptible heme proteins. CO release was investigated in the myoglobin binding assay and found to be rapid (<5 min) with CORM-2- and CORM-3, whereas CORM-401 continuously provided CO (>50 min). Storage stability of CORM stock solutions was also assessed with the myoglobin assay. Only CORM-401 stock solutions were stable over a period of 7 days. Incubation of CORMs with recombinant cytochrome P450 led to an inhibition of enzyme activity. However, only CORM-3 and CORM-401 proved to be suitable in this test system because controls with the inactivated CORM-2 (iCORM-2) also led to a loss of enzyme activity. The impact of CORMs on the respiratory chain was investigated with high resolution respirometry and extracellular flux technology. In the first approach interferences of CORM-2 and CORM-3 with oxygen measurement occurred, since a rapid depletion of oxygen was detected in the medium even when no cells were present. However, CORM-401 did not interfere with oxygen measurement and the expected inhibition of cellular respiration was observed. CORM-2 was not suitable for use in oxygen measurements with the extracellular flux technology and CORM-3 application did not show any effect in this system. However, CO-dependent inhibition of cellular respiration was observed with CORM-401. Based on the present experiments it is concluded, that CORM-401 produced most reliable CO-specific results for the modulation of typical CO targets. For studies on CO-dependent biological effects on intracellular heme groups, CORM-2 and CORM-3 were less suitable. Depending on the experimental setting, data achieved with these compounds should be evaluated with caution.