• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1999년도 학술발표회 진행표 및 논문초록
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• pp.24-25
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• 1999

Intracellular pH(pH$_{i}$) regulation is very important to regulate the cellular functions of cardiac myocytes such as contractility, signal transduction, ion regulation, cell volume, and energy production etc. The resting pH$_{i}$ was maintained at about 7.07 and strictly regulated within the range of $\pm$0.1.(omitted)ted)

• 미생물학회지
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• 제38권3호
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• pp.168-172
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• 2002

출아효모는 주변 환경 pH의 커다란 변화 속에서 적응할 수 있는 효과적인 체계를 지니고 있으며 SHC1 유전자는 알칼리 pH 조건에서 세포의 성장에 필요한 유전자 중에 하나임을 확인하였다. SHC1 유전자의 세포내 pH 조절 기작을 보다 구체적으로 알아보기 위하여 이 유전자가 소실된 돌연변이주를 제조하였다. 성장률의 차이가 나타나는 원인을 세포 내부의 pH 완충능력 결여에 의한 것으로 추측하고 pH 감수성 형광물질인 C.SNARE를 사용하여 외부 pH의 변화에 따른 세포 내부의 pH를 측정하였다. 알칼리 pH 완충효과는 소실 돌연변이의 경우는 야생종 대비 70% 수준을 보였다. 또한 pH 조절에 관여하는 효모세포 내부의 $Na^{+}$과 $K^{+}$의 농도를 원자흡광계를 사용하여 조사한 바, $K^{+}$ 이온의 경우에는 돌연변이주에 비하여 야생형 세포내에 더 많이 존재하는 것으로 나타났으나 $Na^{+}$ 이온의 경우는 별다른 차이점을 보이지 않았다. 이러한 결과는 $K^{+}$ 이온의 조절이 효모에서 세포내 pH조절 기작에 중요하며 SHC1 유전자는 이 $K^{+}$ 이온의 세포내 농도 유지에 관여하고 있다는 것을 제시해 주었다.

• The Korean Journal of Physiology and Pharmacology
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• 제7권3호
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• pp.187-191
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• 2003

$Na^+/H^+$ exchanger (NHE) has a critical role in regulation of intracellular pH (pHi) in the renal proximal tubular cells. It has recently been shown that dopamine inhibits NHE in the renal proximal tubules. Nevertheless, there is a dearth of information on the effects of long-term (chronic) dopamine treatment on NHE activities. This study was performed to elucidate the pHi regulatory mechanisms during the chronic dopamine treatments in renal proximal tubular OK cells. The resting pHi was greatly decreased by chronic dopamine treatments. The initial rate and the amplitude of intracellular acidification by isosmotical $Na^+$ removal from the bath medium in chronically dopamine-treated cells were much smaller than those in control. Although it seemed to be attenuated in $Na^+$-dependent pH regulation system, $Na^+$-dependent pHi recovery by NHE after intracelluar acid loading in the dopamine-treated groups was not significantly different from the control. The result is interpreted to be due to the balance between the stimulation effects of lower pHi on the NHE activity and counterbalance by dopamine. Our data strongly suggested that chronic dopamine treatment increased intrinsic intracellular buffer capacity, since higher buffer capacity was induced by lower resting pHi and this effect could attenuate pHi changes under extracellular $Na^+$-free conditions in chronically dopamine-treated cells. Our study also demonstrated that intracellular acidification induced by chronic dopamine treatments was not mediated by changes in NHE activity.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.57-57
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• 2001

Intracellular pH(pH$\sub$i/) is strictly regulated since it is related to various cellular events such as contractility, signal transduction, ion regulation, cell volume, and energy production etc. In physiological conditions, pH$\sub$i/ of arteriolar smooth muscle faced substantial pressure to be changed during the regulation of blood flow. Therefore it is very important to know the regulatory mechanism of pH$\sub$i/.(omitted)

• Molecules and Cells
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• 제39권1호
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• pp.46-52
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• 2016

It is increasingly apparent that nature evolved peroxiredoxins not only as $H_2O_2$ scavengers but also as highly sensitive $H_2O_2$ sensors and signal transducers. Here we ask whether the $H_2O_2$ sensing role of Prx can be exploited to develop probes that allow to monitor intracellular $H_2O_2$ levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular $H_2O_2$ concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of $H_2O_2$ levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redoxsensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for $H_2O_2$ probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular $H_2O_2$ homeostasis and Prx signaling.

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• 대한소아치과학회지
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• 제25권2호
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• pp.352-367
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• 1998

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

• IMMUNE NETWORK
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• 제3권4호
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• pp.268-275
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• 2003

Background: Hemopoietic cells require the constant presence of growth factors for survival in vitro and in vivo. Caspases have been known as central executors of apoptotic cell death. We have, therefore, investigated the pathways that regulate caspase activity and apoptosis using the $CD34^+$ cell line, TF-1 which requires GM-CSF for survival. Methods: Apoptosis was measured by annexin V staining and mitochondrial membrane potential was measured by DiOC6 labelling. Intracellular pH was measured using pH sensitive fluorochrome, BCECF or SNARF-1, followed by flow cytometry analysis. Caspase activation was analyzed by PARP cleavage using anti-PARP antibody. Results: Removal of GM-CSF induceed PARP cleavage, a hallmark of caspase activity, concomitant with pHi acidification and a drop in mitochondrial potential. Treatment with ZVAD, a competitive inhibitor of caspases, partially rescued cell death without affecting pHi acidification and the reduction of mitochondrial potential, suggesting that both these events act upstream of caspases. Overexpression of Bcl-2 prevented cell death induced by GM-CSF deprivation as well as pHi acidification and the reduction in mitochondrial membrane potential. In parental cells maintained with GM-CSF, EIPA, a competitive inhibitor of $Na^+/H^+$ antiporter induced apoptosis, accompanied by a drastic reduction in mitochondrial potential. In contrast, EIPA induced apoptosis in Bcl-2 transfectants without causing mitochondrial membrane depolarization. Conclusion: Taken together, our results suggest that the regulation of $H^+$fluxes, either through a mitochondriondependent or independent pathway, is central to caspase activation and apoptosis.

• The Korean Journal of Physiology and Pharmacology
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• 제20권4호
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• pp.433-440
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• 2016

Inositol-1,4,5-triphosphate [$IP_3$] receptors binding protein released with $IP_3$ (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

• 한국동물생명공학회지
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• 제35권4호
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• pp.357-365
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• 2020

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.