Oikawa Shino, Kai Yuko, Mano Asuka, Nakamura Shigeo, Kakinuma Yoshihiko
Department of Bioregulatory Science (Physiology), Nippon Medical School Graduate School of Medicine, Toyko, Japan.
Department of Chemistry, Nippon Medical School, Tokyo, Japan.
Cell Physiol Biochem. 2019;52(4):922-934. doi: 10.33594/000000064.
BACKGROUND/AIMS: In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS.
To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO₂ and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system.
Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group.
SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.
背景/目的：在之前的一项研究中，我们报道心肌细胞配备有非神经元性心脏胆碱能系统（NNCCS）以合成乙酰胆碱（ACh），这对于维持心脏的基本生理功能必不可少。本研究的目的是鉴定并表征NNCCS的一种药理学诱导剂。
为了鉴定NNCCS的一种药理学诱导剂，我们筛选了几种化学结构与S-亚硝基-N-乙酰-DL-青霉胺（SNAP）结构相似的化合物。初步研究表明，SNAP是一种非神经元性ACh合成的诱导剂。我们使用蛋白质免疫印迹分析、荧光素酶测定以及细胞内cGMP、NO₂和ACh水平的测量，在H9c2和HEK293细胞中筛选潜在的药理学诱导剂。还使用心脏导管系统评估了筛选出的化合物对雄性C57BL6小鼠心脏功能的影响。
在测试的化合物中，我们选择了S-亚硝基-N-新戊酰-D-青霉胺（SNPiP），它能使H9c2和HEK293细胞中的细胞内cGMP水平和一氧化氮（NO）水平逐渐升高。这些升高的水平导致胆碱乙酰转移酶基因的逐渐反式激活和翻译。此外，体外和体内SNPiP处理使ACh水平在72小时内升高。经SNPiP处理的小鼠心脏功能上调，无心动过速，但舒张功能增强，从而改善心输出量。正如缺乏亚硝基的N-新戊酰-D-青霉胺（PiP）无效所证实的那样，SNPiP的作用依赖于SNPiP的亚硝基基团。
SNPiP被确定为诱导NNCCS的重要药理学候选物之一。
