• The Korean Journal of Physiology and Pharmacology
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• 제23권5호
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• pp.345-356
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• 2019

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, $100{\mu}M$) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to $50{\mu}M$. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated $Ca^{2+}$-transient. This effect was preserved in the presence of BAPTA (10 and $20{\mu}M$) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane ($75{\mu}M$) to inhibit store-operated calcium channel or thapsigargin ($2{\mu}M$) to delete calcium store. In addition, DHA ($12{\mu}M$) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a $Ca^{2+}$ activated mode to a preferentially controlled phosphorylation mode.

• Molecules and Cells
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• 제45권8호
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• pp.575-587
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• 2022

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

• Molecules and Cells
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• 제41권12호
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• pp.1000-1007
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• 2018

Mitochondria and endoplasmic reticulum (ER) are essential organelles in eukaryotic cells, which play key roles in various biological pathways. Mitochondria are responsible for ATP production, maintenance of $Ca^{2+}$ homeostasis and regulation of apoptosis, while ER is involved in protein folding, lipid metabolism as well as $Ca^{2+}$ homeostasis. These organelles have their own functions, but they also communicate via mitochondrial-associated ER membrane (MAM) to provide another level of regulations in energy production, lipid process, $Ca^{2+}$ buffering, and apoptosis. Hence, defects in MAM alter cell survival and death. Here, we review components forming the molecular junctions of MAM and how MAM regulates cellular functions. Furthermore, we discuss the effects of impaired ER-mitochondrial communication in various neurodegenerative diseases.

• Biomolecules & Therapeutics
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• 제32권2호
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• pp.171-182
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• 2024

All cells are equipped with intricate signaling networks to meet the energy demands and respond to the nutrient availability in the body. AMP-activated protein kinase (AMPK) is among the most potent regulators of cellular energy balance. Under ATP -deprived conditions, AMPK phosphorylates substrates and affects various biological processes, such as lipid/glucose metabolism and protein synthesis. These actions further affect the cell growth, death, and functions, altering the cellular outcomes in energy-restricted environments. AMPK plays vital roles in maintaining good health. AMPK dysfunction is observed in various chronic diseases, making it a promising target for preventing and alleviating such diseases. Herein, we highlight the different AMPK functions, especially in allergy, aging, and cancer, to facilitate the development of new therapeutic approaches in the future.

• 한국환경보건학회지
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• 제27권2호
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• pp.82-91
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• 2001

This study was carried out to elucidate the protective effect of zinc chloride(ZnCl$_2$) and its mechanism against the immuno-cytotoxicity of methylmercury chloide($CH_3$HgCl). This study was observed in the culture of EMT-6 cells which are originated from mammary adenocarcinoma of Balb/c mouse. Cytotoxicity of metals was measured by cell viability and NO$_2$$^{[-10]}$ , and mitochondrial function was evaluated by adenosine triphosohate (ATP) production. $CH_3$HgCl significantly decreased the sythesis of nitric oxide(NO), ATP and glutathione(GSH) in a dose-dependent manner. ZnCl$_2$ significantly increased the synthesis of GSH in a dose-dependent manner, but synthesis of NO and ATP were not changed. The immuno-cytotoxicity of $CH_3$HgCl was not fully protected when combined addition of ZnCl$_2$, whereas ZnCl$_2$ prior to addition of $CH_3$HgCl completly protected the Hg-induced immuno-cytotoxicity. Similarly, intracellular accumulation of mercury significantly decreased by ZnCl$_2$. Degree of diminution of intracellular mercury was larger in ZnCl$_2$ prior to addition of $CH_3$HgCl than in combined addition of ZnCl$_2$ and $CH_3$HgCl.. Dithiothreitol(DTT) or buthionine sulfoximine(BSO) addition at 50$\mu$M or less, which was not toxic to the cells, did not affect synthesis of NO and ATP. DTT increased intracellular GSH level and DTT pretreatment protected toxicity induced by $CH_3$HgCl as shown complete recover in the NO and ATP values. BSO decreased intracellular GSH level and BSO pretreatment exaggerated toxicity induced by $CH_3$HgCl as shown synergistic reduction in the NO and ATP values. These results indicated that the protective effects of zinc against immuno-cytotoxicity of methylmercury associated with increasing cellular level of GSH. Increased intracellular GSH transports methylmercury to out of cells. In accordance with intracellular level of mercury decreased, immuno-cytotoxicity of methylmercury decreased. These result also suggest that the protective mechanism of zinc against the mercury toxicity would be exerted in the immune system in vivo.

• 대한임상검사과학회지
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• 제53권2호
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• pp.158-164
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• 2021

고중성지방혈증은 죽상동맥경화증의 주요한 위험 요인 중 하나이다. 중성지방은 대식세포의 세포 사멸을 유도하여 죽상동맥경화증 발생에 기여하는 것으로 알려져 있다. 본 연구팀은 앞선 연구에서 대식세포의 중성지방-유도 세포 사멸이 pannexin-1 활성화에 의한 세포 외 ATP 농도 증가, caspase-2와 caspase-1 활성화, caspase-8을 포함한 apoptotic caspase 활성화 경로로 일어나는 것을 보고하였다. 한편 다른 연구들에서는 세포 내 다른 여러 기전에서 caspase-8이 caspase-1과 -2의 상위 단백질이라 보고하고 있다. 따라서 본 연구에서는 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 상위단백지로 영향을 미치는지 여부를 조사하기 위해 수행되었다. 본 연구진은 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 caspase-3 활성화 및 PARP 절단을 유도하였다. 다음으로 중성지방이 처리된 대식세포에서 caspase-8 억제 시, caspase-8의 상위 단백질로 보고한 caspase-1 및 -2의 활성이 감소하는 것을 확인하였다. 또한 ATP 처리 시 caspase-8 억제제 처리에 의해 감소된 caspase-2의 활성이 회복되는 것을 확인하였다. 위의 결과를 통해 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 세포 외부 ATP 농도 증가에 관여하는 단백질 또는 그 상위 기전에 양성피드백 방식으로 영향을 미쳐 caspase-1과 -2를 활성화하여 중성지방-유도 대식세포 사멸을 증진시킴을 알 수 있다.

• Molecules and Cells
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• 제28권3호
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• pp.139-148
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• 2009

Cell death has been traditionally classified in apoptosis and necrosis. Apoptosis, known as programmed cell death, is an active form of cell death mechanism that is tightly regulated by multiple cellular signaling pathways and requires ATP for its appropriate process. Apoptotic death plays essential roles for successful development and maintenance of normal cellular homeostasis in mammalian. In contrast to apoptosis, necrosis is classically considered as a passive cell death process that occurs rather by accident in disastrous conditions, is not required for energy and eventually induces inflammation. Regardless of different characteristics between apoptosis and necrosis, it has been well defined that both are responsible for a wide range of human diseases. Glycogen storage disease type I (GSD-I) is a kind of human genetic disorders and is caused by the deficiency of a microsomal protein, glucose-6-phosphatase-${\alpha}$ ($G6Pase-{\alpha}$) or glucose-6-phosphate transporter (G6PT) responsible for glucose homeostasis, leading to GSD-Ia or GSD-Ib, respectively. This review summarizes cell deaths in GSD-I and mostly focuses on current knowledge of the neutrophil apoptosis in GSD-Ib based upon ER stress and redox signaling.

• The Korean Journal of Physiology and Pharmacology
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• 제23권1호
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• pp.37-45
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• 2019

To study the effect of nicorandil pretreatment on ketone body metabolism and Acetyl-CoA acetyltransferase (ACAT1) activity in hypoxia/reoxygenation (H/R)-induced cardiomyocytes. In our study, we applied H9c2 cardiomyocytes cell line to evaluate the cardioprotective effects of nicorandil. We detected mitochondrial viability, cellular apoptosis, reactive oxygen species (ROS) production and calcium overloading in H9c2 cells that exposed to H/R-induced cytotoxicity. Then we evaluated whether nicorandil possibly regulated ketone body, mainly ${\beta}$-hydroxybutyrate (BHB) and acetoacetate (ACAC), metabolism by regulating ACAT1 and Succinyl-CoA:3-ketoacid coenzyme A transferase 1 (OXCT1) protein and gene expressions. Nicorandil protected H9c2 cardiomyocytes against H/R-induced cytotoxicity dose-dependently by mitochondria-mediated anti-apoptosis pathway. Nicorandil significantly decreased cellular apoptotic rate and enhanced the ratio of Bcl-2/Bax expressions. Further, nicorandil decreased the production of ROS and alleviated calcium overloading in H/R-induced H9c2 cells. In crucial, nicorandil upregulated ACAT1 and OXCT1 protein expressions and either of their gene expressions, contributing to increased production of cellular BHB and ACAC. Nicorandil alleviated cardiomyocytes H/R-induced cytotoxicity through upregulating ACAT1/OXCT1 activity and ketone body metabolism, which might be a potential mechanism for emerging study of nicorandil and other $K_{ATP}$ channel openers.

• Molecules and Cells
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• 제46권11호
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• pp.655-663
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• 2023

Autophagy dysfunction is associated with human diseases and conditions including neurodegenerative diseases, metabolic issues, and chronic infections. Additionally, the decline in autophagic activity contributes to tissue and organ dysfunction and aging-related diseases. Several factors, such as down-regulation of autophagy components and activators, oxidative damage, microinflammation, and impaired autophagy flux, are linked to autophagy decline. An autophagy flux impairment (AFI) has been implicated in neurological disorders and in certain other pathological conditions. Here, to enhance our understanding of AFI, we conducted a comprehensive literature review of findings derived from two well-studied cellular stress models: glucose deprivation and replicative senescence. Glucose deprivation is a condition in which cells heavily rely on oxidative phosphorylation for ATP generation. Autophagy is activated, but its flux is hindered at the autolysis step, primarily due to an impairment of lysosomal acidity. Cells undergoing replicative senescence also experience AFI, which is also known to be caused by lysosomal acidity failure. Both glucose deprivation and replicative senescence elevate levels of reactive oxygen species (ROS), affecting lysosomal acidification. Mitochondrial alterations play a crucial role in elevating ROS generation and reducing lysosomal acidity, highlighting their association with autophagy dysfunction and disease conditions. This paper delves into the underlying molecular and cellular pathways of AFI in glucose-deprived cells, providing insights into potential strategies for managing AFI that is driven by lysosomal acidity failure. Furthermore, the investigation on the roles of mitochondrial dysfunction sheds light on the potential effectiveness of modulating mitochondrial function to overcome AFI, offering new possibilities for therapeutic interventions.

• 동의생리병리학회지
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• 제16권3호
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• pp.521-527
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• 2002

The Caesalpinia sappan is widely used in the traditional oriental herbal medicine for anti-inflammatory, antioxidant effects. The effects of water extract of C. sappan on the cell viability and induction of apoptosis were investigated in human lung cancer cell line A549. The water extract of C. sappan produced apoptotic cell death and DNA fragmentation and nucleus chromatin condensation in A549 cells. The enzyme activity of caspase-3 and protein level of actived caspase-3 were markedly increased in A549 cells treated with the water extract of C. sappan. In addition, the extract of C. sappan induced cleavage of Poly (ADP-ribose) polymerase (PARP), a known substrate for caspase-3, and dropped in cellular ATP levels. These results suggest that the extract of C. sappan exerts anticancer activity by induction of apoptosis via activation of caspase-3, cleavage of PARP protein, and depletion of cellular ATP levels in A549 cells.