Department of Physiology, China Medical University, Taichung 40402, Taiwan.
Department of Internal Medicine, Gastroenterology and Hepatology Section, An Nan Hospital, China Medical University, Tainan, Taiwan.
Neurotoxicology. 2023 Sep;98:9-15. doi: 10.1016/j.neuro.2023.07.001. Epub 2023 Jul 8.
Activity or expression of sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA) is diminished in some disease states such as cardiac failure and diabetes mellitus. A newly developed activator of SERCA, CDN1163, reportedly rescued or alleviated pathological conditions attributed to dysfunctional SERCA. We examined whether CDN1163 could relieve mouse neuronal N2A cell growth inhibition caused by cyclopiazonic acid (CPA, SERCA inhibitor). We also examined how CDN1163 affected cytosolic Ca, mitochondrial Ca and mitochondrial membrane potential.
Cell viability was measured by MTT assay and trypan blue exclusion test. Cytosolic Ca, mitochondrial Ca and mitochondrial membrane potential were measured using fura 2, Rhod-2 and JC-1, respectively, as fluorescent probes.
CDN1163 (10 μM) itself suppressed cell proliferation, and did not alleviate CPA's inhibitory effect (and vice versa). Cell cycle was arrested at the G1 phase after CDN1163 treatment. CDN1163 treatment caused a slow yet persistent cytosolic [Ca] elevation partly due to Ca release from an internal store other than the CPA-sensitive endoplasmic reticulum (ER). Treatment with CDN1163 for 3 h raised mitochondrial Ca level and such increase was suppressed by MCU-i4 (an inhibitor of mitochondria Ca uniporter, MCU), suggesting Ca entered the mitochondrial matrix through MCU. Treatment of cells with CDN1163 up to 2 days resulted in mitochondrial hyperpolarization.
CDN1163 caused internal Ca leak, cytosolic Ca overload, mitochondrial Ca elevation and hyperpolarization, cell cycle arrest and cell growth inhibition.
在某些疾病状态下，如心力衰竭和糖尿病，肌浆/内质网 Ca2+-ATP 酶（SERCA）的活性或表达减少。一种新开发的 SERCA 激活剂 CDN1163，据报道可以挽救或缓解由于功能失调的 SERCA 引起的病理状况。我们研究了 CDN1163 是否可以缓解环匹阿尼酸（CPA，SERCA 抑制剂）引起的小鼠神经元 N2A 细胞生长抑制。我们还研究了 CDN1163 如何影响细胞溶质 Ca、线粒体 Ca 和线粒体膜电位。
通过 MTT 测定法和台盼蓝排斥试验测定细胞活力。使用荧光探针 fura 2、Rhod-2 和 JC-1 分别测量细胞溶质 Ca、线粒体 Ca 和线粒体膜电位。
CDN1163（10 μM）本身抑制细胞增殖，并且不能缓解 CPA 的抑制作用（反之亦然）。CDN1163 处理后，细胞周期被阻滞在 G1 期。CDN1163 处理导致细胞溶质 [Ca]缓慢而持续升高，部分原因是除了 CPA 敏感的内质网（ER）之外，从内部储存库释放 Ca。用 MCU-i4（线粒体 Ca 单向转运蛋白 MCU 的抑制剂）处理 3 h 可提高线粒体 Ca 水平，这种增加被抑制，表明 Ca 通过 MCU 进入线粒体基质。用 CDN1163 处理细胞长达 2 天导致线粒体超极化。
CDN1163 引起内部 Ca 泄漏、细胞溶质 Ca 过载、线粒体 Ca 升高和超极化、细胞周期停滞和细胞生长抑制。
