• The Korean Journal of Physiology and Pharmacology
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• v.22 no.3
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• pp.311-319
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• 2018

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (${\Delta}{\psi}_m$). Therefore, pharmacological manipulation of ${\Delta}{\psi}_m$ can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ${\Delta}{\psi}_m$ against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity ($100{\mu}M$, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate ($100{\mu}M$)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of $Ca^{2+}$ ($5{\mu}M$). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ${\Delta}{\psi}_m$ were completely abolished in $K^+-free$ medium on pure isolated mitochondria. Taken together, results demonstrate that $K^+$ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial $K^+$ influx is probably mediated, at least in part, by activation of mitochondrial $K^+$ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.515-525
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• 2022

Background: The incidence of ischemic cerebrovascular disease is increasing in recent years and has been one of the leading causes of neurological dysfunction and death. Ginsenoside Rg1 has been found to protect against neuronal damage in many neurodegenerative diseases. However, the effect and mechanism by which Rg1 protects against cerebral ischemia-reperfusion injury (CIRI) are not fully understood. Here, we report the neuroprotective effects of Rg1 treatment on CIRI and its possible mechanisms in mice. Methods: A bilateral common carotid artery ligation was used to establish a chronic CIRI model in mice. HT22 cells were treated with Rg1 after OGD/R to study its effect on [Ca²⁺]_i. The open-field test and poleclimbing experiment were used to detect behavioral injury. The laser speckle blood flowmeter was used to measure brain blood flow. The Nissl and H&E staining were used to examine the neuronal damage. The Western blotting was used to examine MAP2, PSD95, Tau, p-Tau, NOX2, PLC, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging was used to test the level of [Ca²⁺]_i. Results: Rg1 treatment significantly improved cerebral blood flow, locomotion, and limb coordination, reduced ROS production, increased MAP2 and PSD95 expression, and decreased p-Tau, NOX2, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging results showed that Rg1 could inhibit calcium overload and resist the imbalance of calcium homeostasis after OGD/R in HT22 cells. Conclusion: Rg1 plays a neuroprotective role in attenuating CIRI by inhibiting oxidative stress, calcium overload, and neuroinflammation.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.6
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• pp.653-659
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• 1998

• Journal of Nutrition and Health
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• v.32 no.3
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• pp.248-258
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• 1999

This study examined the effect of excess loading of calcium (Ca)and iron(Fe) on the bioavailability of minerals in both normal and Ca-and Fe-deficient rats. Three-week-old male rats were divided into four groups and fed experimental diets for six weeks, containing either normal (0.5%) or high(1.5%) Ca and normal (35ppm) or high (350ppm)Fe. Likewise, three-week-old male rats were first fed a Ca-and Fe-deficient diet for three weeks, and then fed one of four experimental diets for additional three weeks. In both normal and Ca-and Fe-deficient rats, ca contents of serum, liver, kidney and femur were not significantly affected by dietary Ca and Fe levels. Apparent Ca absorption(%) decreased in rats fed a high Ca diet regardless of dietary Fe levels. Magnesium(Mg) contents of serum, liver and femur significantly decreased in rats fed a high Ca diet. Fe contents of serum and liver significantly increased in rats fed a high-Fe diet, but decreased in rats fed a high Ca diet. Fe content of serum and liver significantly increased in rate fed a high-Fe diet, decreased in rats fed a high-Ca diet. Apparent Fe absorption increased in rats fed a high-Fe diet, and decreased in rats fed a high-Ca diet in Ca-and Fe-deficient rats, but dietary Ca did not seem to affect Fe absorption in normal rats. Phosphorus(P) contents of serum and femur were not significantly affected by dietary Ca and Fe levels in both normal and Ca-and Fe-deficient rats. Serum copper(Cu) decreased in rats fed a high-Fe diet, while serum zinc(Zn) decreased in rats fed a high-Ca diet in normal rats. Cu contents of liver, and Zn contents of serum and liver decreased in rats fed a high-Fe diet in Ca-and Fe-deficient rats. There results suggest that a dietary overload of Ca and Fe in both normal and Ca-and Fe-deficient rats may decrease mineral bioavailability leading to potential health problems.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.5
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• pp.537-546
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• 1997

It has been postulated that the intracellular taurine is co-transported with $Na^+$down a concentration gradient and prevents the intracellular accumulation of sodium. It is therefore, expected that an elevated level of intracellular taurine prevents the sodium-promoted calcium influx to protect the cellular damages associated with sodium and calcium overload. In the present study, we evaluated the effects of intra- and extracellular taurine on the myocardial $Na^+$and$Ca^{++}$ contents and the cardiac functions in isolated rat hearts which were loaded with sodium by monensin, a $Na^+-ionophore$. Monensin caused a dose-dependent increase in intracellular $Na^+$ accompanied with a subsequent increase in intracellular $Ca^{++}$ and a mechanical dysfunction. In this monensin-treated heart, myocardial taurine content was decreased with a concomittent increase in the release of taurine. The monensin-induced increases in intracellular $Na^+$, $Ca^{++}$ and depression of cardiac function were prevented in the hearts of which taurine content had been increased by high-taurine diet. Conversely, in the hearts of which taurine concentration gradient had been decreased by addition of taurine in the perfusate, the monensin-induced increases in $Na^+$, $Ca^{++}$ and functional depression were accelerated. These results suggest that taurine, depending on the intra-extracellular concentration gradient, can affect intracellular sodium and calcium concentrations, and that an increased intracellular taurine may play a role in protection of myocardial dysfunction associated with the sodium and calcium overload.

• BMB Reports
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• v.54 no.6
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• pp.305-310
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• 2021

Cereblon (CRBN) is a multi-functional protein that acts as a substrate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells.

• Animal cells and systems
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• v.8 no.2
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• pp.97-103
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• 2004

The cytotoxic effect of iron was examined in peritoneal macrophage to determine contributing factors by iron injection to rat. Viability was reduced by 24% by the iron-overload and by 30% by short-term iron addition. Total iron was increased by 45% in the iron-overloaded with remarkable elevation (9 to 14 fold) in the presence of $FeSO_4$. Free calcium was also increased by 19% in control and 44% in iron-overloaded group due to additional $FeSO_4$ NO and MDA were increased by 40% and 136%, respectively, with significant reduction (37%) of NAD(P)H. RCR and cytochrome c oxidase activity were lowered approximately by 10% with reduction of mitochondrial membrane potential. Addition of iron was frequently associated with altered distribution of mitochondria of high membrane potential in the iron-overloaded macrophage. These results suggest altered mitochondria with high NO and low NAD(P)H due to iron.

• Animal cells and systems
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• v.14 no.2
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• pp.91-98
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• 2010

Male rats were given a single injection of iron, and temporal changes in iron content and iron-induced effects were examined in heart cellular fractions. Over a period of 72 h, the contents of total and labile iron, reactive oxygen species, and NO in tissue homogenate, nuclear debris, and postmitochondrial fractions were mostly constant, but in mitochondria they continuously increased. An abrupt decrease in membrane potential and NAD(P)H at 12 h was also found in mitochondria. The respiratory control ratio was reduced slowly with a slight recovery at 72 h, suggesting uncoupling by iron.While the ATP content of tissue homogenate decreased steadily until 72 h, it showed a prominent increase in mitochondria at 12 h. Total iron and calcium concentration also progressively increased in mitochondria over 72 h. Enzyme activity of the oxidative phosphorylation system was significantly altered by iron injection: activities of complexes I, III, and IV were reduced considerably, but complex II activity and the ATPase activity of complex V were enhanced. A reversal of activity in complexes I and II at 12 h suggested reverse electron transfer due to iron overload. These results support the argument that mitochondrial activities including oxidative phosphorylation are modulated by excessive iron.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.1
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• pp.39-48
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• 2023

Spinal nerve injury causes mechanical allodynia and structural imbalance of neurotransmission, which were typically associated with calcium overload. Storeoperated calcium entry (SOCE) is considered crucial elements-mediating intracellular calcium homeostasis, ion channel activity, and synaptic plasticity. However, the underlying mechanism of SOCE in mediating neuronal transmitter release and synaptic transmission remains ambiguous in neuropathic pain. Neuropathic rats were operated by spinal nerve ligations. Neurotransmissions were assessed by whole-cell recording in substantia gelatinosa. Immunofluorescence staining of STIM1 with neuronal and glial biomarkers in the spinal dorsal horn. The endoplasmic reticulum stress level was estimated from qRT-PCR. Intrathecal injection of SOCE antagonist SKF96365 dose-dependently alleviated mechanical allodynia in ipsilateral hind paws of neuropathic rats with ED₅₀ of 18 ㎍. Immunofluorescence staining demonstrated that STIM1 was specifically and significantly expressed in neurons but not astrocytes and microglia in the spinal dorsal horn. Bath application of SKF96365 inhibited enhanced miniature excitatory postsynaptic currents in a dosage-dependent manner without affecting miniature inhibitory postsynaptic currents. Mal-adaption of SOCE was commonly related to endoplasmic reticulum (ER) stress in the central nervous system. SKF96365 markedly suppressed ER stress levels by alleviating mRNA expression of C/ EBP homologous protein and heat shock protein 70 in neuropathic rats. Our findings suggested that nerve injury might promote SOCE-mediated calcium levels, resulting in long-term imbalance of spinal synaptic transmission and behavioral sensitization, SKF96365 produces antinociception by alleviating glutamatergic transmission and ER stress. This work demonstrated the involvement of SOCE in neuropathic pain, implying that SOCE might be a potential target for pain management.

• Biomolecules & Therapeutics
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• v.16 no.1
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• pp.21-27
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• 2008

Clotrimazole (CLT), a potent antifungal drug, is known to inhibit tumor cell proliferation. In the present study, we examined the role of intracellular $Ca^{2+}$ in CLT-induced cell cycle arrest of colon adenocarcinoma HT29 cells. CLT inhibited growth of HT29 cells in a concentration-dependent manner, which was associated with inhibition of cell cycle progression at the G(1)-S phase transition and an increase in the expression of cell cycle inhibitor proteins p27 and p53. CLT also suppressed the $Ca^{2+}$ overload by A23187, a calcium ionophore, suggesting its role in modulation of intracellular $Ca^{2+}$ concentration in HT29 cells. The simultaneous application of CLT and A23187 with addition of $CaCl_2$ (1mM) to the medium significantly reversed CLT-induced p27 and p53 protein level increase and growth suppression. Our results suggest that CLT induces cell cycle arrest of colon adenocarcinoma HT29 cells via induction of p27 and p53, which may, at least in part, be mediated by alteration of intracellular $Ca^{2+}$ level.