• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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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.

• 대한생식의학회:학술대회논문집
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• 2002.11a
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• pp.94-95
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• 2002

• Archives of Pharmacal Research
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• v.29 no.8
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• pp.657-665
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• 2006

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and $Ca^{2+}$ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and $Ca^{2+}$ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and $Ca^{2+}$, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and $Ca^{2+}$-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

• Molecules and Cells
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• v.23 no.1
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• pp.11-16
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• 2007

N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular $Ca^{2+}$ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular $Ca^{2+}$ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the $G_{i/o}$ protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular $Ca^{2+}$ with the $Ca^{2+}$ chelator EGTA or depletion of intracellular $Ca^{2+}$ stores with thapsigargin impeded the DMS-induced increase of intracellular $Ca^{2+}$ concentration. Pretreatment of cells with $NH_4Cl$ or monensin reduced the DMS-induced $Ca^{2+}$ increase. However, inhibition of the DMS-induced $Ca^{2+}$ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular $Ca^{2+}$ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.

• Proceedings of the Korean Biophysical Society Conference
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• 2001.06a
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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)

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.432-438
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• 1995

The intracellular alginase from Vibrio sp. AL-145 was purified by ion chromatography on DEAE-Cellulose column, Q-Sepharose column, and gel filtration on Sephadex G-100 column. The optimum pH and temperature for the activity of the purified intracellular enzyme were 8.0 and 37$\circ$C, respectively. The enzyme was stable at the pH range of 7.5-8.5, and at 30$\circ$C for 30 min. The molecular weight of the intracellular enzyme was estimated to be about 23, 000 daltons by SDS-polyacrylamide gel electrophoresis. NaCl was required for enzyme activity and the optimum concentration was 0.5 M. The activity of intracellular enzyme was inhibited by Co$^{2+}$, Hg$^{2+}$, Zn$^{2+}$, 0-phenanthroline, $\rho$-CMB, EDTA and iodoacetate, and stimulated by Ca$^{2+}$, L-cysteine and 2-mercaptoethanol. This enzyme was an alginase specifically degrading alginic acid.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.9
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• pp.1143-1150
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• 2017

The aim of this study was to determine the chemical and intracellular antioxidant activities of ${\beta}$-carotene and lycopene and to compare their quantitative structure-activity relationship (QSAR). In our previous study, the second ionization energy of lycopene was higher than that of ${\beta}$-carotene, as calculated by QSAR. Chemical antioxidant activities of ${\beta}$-carotene, lycopene, and Trolox were examined by measuring ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Intracellular antioxidant activities were evaluated by intracellular reactive oxygen species (ROS) and DNA fragmentation. The FRAP of lycopene was higher than that of ${\beta}$-carotene (P<0.05), and the two carotenoids had similar antioxidant activities in DPPH radical scavenging activity assay. Trolox had the greatest chemical antioxidant activities (P<0.05). When RAW264.7 cells were treated with lipopolysaccharide (LPS) (100 ng/mL) for 20 h, intracellular ROS and DNA fragmentation significantly increased (P<0.05). RAW 264.7 cells pretreated with ${\beta}$-carotene ($4{\mu}M$) and lycopene ($0.4{\sim}2{\mu}M$) for 4 h formed significantly less intracellular ROS than LPS-treated control cells (P<0.05), whereas cells with Trolox did not reduce production of intracellular ROS. In addition, cells pretreated with $2{\mu}M$ lycopene produced less intracellular ROS than those treated with ${\beta}$-carotene (P<0.05). DNA fragmentation of cells with ${\beta}$-carotene and lycopene was similar to that of LPS-treated control cells as measured by Hoechst staining. The antioxidant ability of lycopene was greater than that of ${\beta}$-carotene in the QSAR, FRAP, and intracellular ROS assays (P<0.05). ${\beta}$-Carotene and lycopene had lower antioxidant activities as measured by FRAP (P<0.05) but higher intracellular protective effects against LPS-induced oxidative stress in comparison with Trolox.

• Korean Journal of Microbiology
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• v.25 no.2
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• pp.110-116
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• 1987

As a pary of investgation on effective accumulation of cadmium in yeast cells, effect of surfactant was studied on intracellular accumulation of cadmium in extremely cadmium-tolerant yeast, Hansenula anomala B-7. Cadmium accumulation was enhanced up to approximately 40% by the addition of non-ionic surfactant, Triton X-100 and its optimal concentration was found to be 0.1-0.2%. Furthermore, optimum conditions for intracellular accumulation of cadmium were at $40^{\circ}C$ and initial pH 6.0 for 48 hours under shaking culture.

• IMMUNE NETWORK
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• v.3 no.4
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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.

• Radiation Oncology Journal
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• v.13 no.2
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• pp.121-128
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• 1995

Purpose: The enhanced cytotoxic effect of combined treatment of hyper-thermia and chemotherapy by increasing intracellular acidity with HMA was investigated. Materials and Methods: FSall tumor cells were injected on the hindlegs of female $C_3H$ mice. When the tumor volume reached about 200mm3, experiments were performed on the groups classified as follows: Group I :Control, Group II : Melphalan alone (2.5mg/kg, 5mg/kg, 10mg/kg, 15mg/kg), Group III : Heat alone $(42.5^{\cdot}C$ for 1 hour) Group IV : Melphalan + Heat $(42.5^{\cdot}C$ for 1 hour), Group V : HMA(10mg/kg) + Melphalan(5.0mg/kg) + Heat$(42.5^{\cdot}C$ for 1hour). Each group included 8-12 mice on each experiment HMA (3-amino-6-chloro-5-(1-homopiperidyl )-N-(diaminomethylene) -c-pyrazinecarboxamide), an analog of amiloride which increases intracellular pH(pHi) was dissolved in dimethyl sulfoxide (DMS) and injected into the tumor-bearing mice through the tail vein. 10mg/kg of HMA and each dose of melphalan were injected into peritoneum of the tumor-bearing mice 30 minutes before heating. Tumor growth delay was calculated when the tumor volme reached at $1500mm^3$ Excision assay was performed on each group and repeated 2-4 times. Results : Tumor growth delay of each experimental groups at $1500mm^3$ were 9, 10, 13 and 19 days respectively. In vivo-in vitro excision assay using FSall tumor cells, the cytotoxicity of each experimental groups was $1.2{\times}10^7,\;1{\times}10^7,\;6{\times}10^6,\;1.7{\times}10^6\;and\;1{\times}10^5$ clonogenic cells/gm respectively When HMA was added to the combined treatment of heat and .chemotherapy, the tumor growth was delayed more than combined treatment without HMA i.e., 6 days tumor growth delay at $1500mm^3$ of tumor volume. Conclusion: The combined effect of cytotoxicity by heat and chemotherapy can be much more enhanced by HMA.