Joohyun Nam - Academia.edu (original) (raw)
Papers by Joohyun Nam
Planta medica, 2015
Cyperus rotundus is used as an analgesic and sedative in oriental medicine and has been reported ... more Cyperus rotundus is used as an analgesic and sedative in oriental medicine and has been reported to exhibit antinociceptive and anti-inflammatory effects. On the other hand, the transient receptor potential vanilloid 1 channel is a nonselective cation channel that senses various noxious chemical and thermal stimuli. However, it has recently been reported that the epidermally expressed transient receptor potential vanilloid 1 channel is involved in heat- and UV-induced skin aging. The aim of this study was to evaluate whether C. rotundus extract and its constituents can inhibit this channel. Ethylacetate and hexane fractions of the methanol extract were found to partially inhibit transient receptor potential vanilloid 1 channel activity, and at a concentration of 90 µM, oleanolic acid, which was one of three constituents isolated from the ethylacetate fraction, inhibited this activity by 61.4 ± 8.0 %. This is first electrophysiological study to be conducted on the effects of C. rotun...
PLoS ONE, 2013
Transthyretin (TTR) is a known carrier protein for thyroxine (T 4) and retinol-binding protein in... more Transthyretin (TTR) is a known carrier protein for thyroxine (T 4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTR kd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOG kd) did not have any effect on TTR gene expression. Both TTR kd and MYOG kd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T 4 uptake during myogenesis was observed in TTR kd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.
Pflugers Arch - Eur J Physiol, 2008
Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we f... more Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we found that WEHI-231, an immature B cell line, expresses voltage-independent K(+) channels called large-conductance background K( + ) channels (LK(bg)). Arachidonic acid (AA) has attracted attention because of its potential regulatory roles in the apoptosis of immature B cells. To elucidate the functional targets of AA, we investigated the effects of AA on membrane currents, voltages, and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) of WEHI-231 and Bal-17 cells that represent immature and mature mouse B cells, respectively. In whole-cell patch clamp, both Kv and IKCa1 were inhibited by AA. On the other hand, AA activated LK(bg) current and non-selective cationic (NSC) current in WEHI-231 while only NSC current in Bal-17. Inside-out patch clamp study showed that AA directly activates LK(bg). AA induced hyperpolarization of WEHI-231 and depolarization of Bal-17 cells, respectively. The selective functional expression of LK(bg) and their activation by AA were also confirmed in the immature B cells (B220(+)/AA4.1(+)) freshly isolated from mouse spleen. In fura-2 spectrofluorimetry, AA induced persistent increase in [Ca(2+)](c) of WEHI-231 cells, which was attenuated by KCl-induced depolarization. In Bal-17 cell, however, AA induced only a transient increase of [Ca(2+)](c). In summary, the novel type of background K(+) channels (LK(bg)) in immature B cells is strongly activated while the other K(+) channels (Kv and IKCa1) commonly expressed in lymphocytes are inhibited by AA. The hyperpolarization and augmentation of Ca(2+) influx by LK(bg) activation might play a role in the response of immature B cells to AA.
Cellular Physiology and Biochemistry, 2014
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCo... more This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2014
The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO2, 2... more The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO2, 2-5% P(O2)). However, functional studies of B cells in hypoxic conditions are sparse. Recently, we reported the expression in mouse B cells of TASK-2, a member of pH-sensitive two-pore domain K(+) channels with background activity. In this study, we investigated the response of K(+) channels to sustained PhyO2 (sustained hypoxia [SH], 3% P(O2) for 24 h) in WEHI-231 mouse B cells. SH induced voltage-independent background K(+) conductance (SH-K(bg)) and hyperpolarized the membrane potential. The pH sensitivity and the single-channel conductance of SH-K(bg) were consistent with those of TASK-2. Immunoblotting assay results showed that SH significantly increased plasma membrane expressions of TASK-2. Conversely, SH failed to induce any current following small interfering (si)TASK-2 transfection. Similar hypoxic upregulation of TASK-2 was also observed in splenic primary B cells. Mechanistica...
The Prostate, 2003
The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neuron... more The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.
The Journal of Physiology, 2007
In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C... more In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP 2) is not known yet. The mouse B cells express large conductance background K + channels (LK bg) that are inhibited by PIP 2. In inside-out patch clamp studies, the application of MgATP (1 mM) also inhibited LK bg due to the generation of PIP 2 by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK bg , which was antagonized by PIP 2 (> 1 μM) or higher concentration of MgATP (5 mM). The inhibition by PIP 2 was partially reversible. However, the application of methyl-β-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK bg activity and facilitated the activation by stretch. In cell-attached patches, LK bg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK bg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LK bg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCγ2 and PIP 2 hydrolysis of plasma membrane. The time dependence of PIP 2 hydrolysis and LK bg activation were similar. The presence of LK bg and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP 2 caused by stretch-activated PLC releases LK bg from the tonic inhibition by PIP 2 .
Proceedings of the National Academy of Sciences, 2013
Sequences meeting the above criteria were further classified by the Ribosomal Database Project (R... more Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.5 using training set 9 (46) from phylum to genus level." should instead appear as "Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.2 using training set 6 (46) from phylum to genus level."
Pflügers Archiv - European Journal of Physiology, 2006
Voltage-dependent, outwardly rectifying anion channels have been described in various cells inclu... more Voltage-dependent, outwardly rectifying anion channels have been described in various cells including lymphocytes. In this study, we found that murine B cells express the voltage-dependent slowly activating anion channels (VSACs). Using a whole-cell configuration, I (VSAC) in Bal-17 was induced by a sustained depolarization (>0 mV) which was remarkably facilitated at 35 degrees C (Q (10)=23 at 30 mV of clamp voltage). Substitution of extracellular Cl(-) with gluconate shifted the reversal potential to the right (35.7 mV). Gd(3+) (IC(50)=0.11 microM) significantly attenuated I (VSAC), but DIDS partially blocked I (VSAC). In addition, extracellular acidification suppressed I (VSAC) whereas alkalinization facilitated the channel activation. I (VSAC) was decreased by 90% at pH 6.35 and increased by 180% at pH 8.0. In cell-attached and inside-out patch clamps, depolarization slowly activated the anion channels of large conductance (approximately 270 pS) with multiple levels of subconductances. The single channel currents were also blocked by Gd(3+) and acidic pH. Furthermore, I (VSAC) was also observed in WEHI-231 (an immature B cell line) and freshly isolated splenic B cells of mice. In summary, murine B cells express unique voltage-dependent anion channels that show a strong sensitivity to both temperature and extracellular pH. Further investigation is required to understand the physiological roles of VSAC and its molecular identity.
Pflügers Archiv - European Journal of Physiology, 2008
K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells... more K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K+ channel (LK bg) in addition to the voltage-gated K+ channel (Kv) and Ca2+-activated K+ channel current (IKCa1). Mibefradil, a blocker of T-type Ca2+ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK bg current with half inhibitory concentration (IC50), 1.9 and 2.3 microM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca2+ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 microM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 microM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1.
Pfl�gers Archiv European Journal of Physiology, 2004
In this study, the short-circuit currents (I sc) of electrolyte absorption and secretion in neona... more In this study, the short-circuit currents (I sc) of electrolyte absorption and secretion in neonatal and adult rat colonic mucosa were compared and the role of Ca 2+ influx through luminal membranes examined in relation to the replenishment of intracellular Ca 2+ stores in colonic crypt cells. Neonatal tissues displayed higher amiloridesensitive I sc and larger increases of electrogenic Cl − secretion in response to an increase in cytosolic [Ca 2+ ] ([Ca 2+ ] c) or cAMP than found in adult colonic epithelium. Ca 2+-mediated Cl − secretion as reflected in the I sc responses to carbachol (I sc,CCh) showed milder "rundown" in neonates than in adult rats. We then employed the relatively stable I sc,CCh of the neonatal colon to investigate the polarity of Ca 2+ entry pathway after muscarinic stimulation. Repetitive stimulation with CCh under Ca 2+-free conditions emptied the intracellular Ca 2+ stores and abolished the I sc,CCh. Re-adding Ca 2+ to the basolateral perfusate rapidly restored I sc,CCh (about 71% of control in 10 min). In contrast, after re-adding Ca 2+ to the luminal perfusate only, the recovery of I sc,CCh took much longer and was incomplete, recovering to only 28% of control after 30 min. Recovery was accelerated by increasing [Ca 2+ ] in the luminal perfusate (5 mM) and blocked by the presence of Gd 3+ (100 µM) in the luminal perfusate. The above results suggest that, in addition to the predominant role of Ca 2+ entry through the basolateral membrane, the influx of Ca 2+ through luminal membranes might also play a role in the Ca 2+ homeostasis of colonic epithelial cells.
Molecules and Cells, 2013
Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transien... more Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca 2+ activated Clchannels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720-P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.
Molecules and Cells, 2010
Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) ar... more Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca(2+)] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca(2+)](c) in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca(2+) entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca(2+)](c) increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca(2+)](c) or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca(2+)](c). Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca(2+) was measured using Ba(2+) as a surrogate ion. Similarly, Ba(2+) influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca(2+)-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca(2+) release was insignificant in primary B cells and inWEHI-231.
Molecules and Cells, 2012
Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and p... more Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and pharmacological actions in various organs. However, little is known about the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker potentials by ginsenoside in the interstitial cells of Cajal (ICCs) using the whole-cell patch clamp technique in the current clamp mode. Among ginsenosides, we investigated the effects of ginsenoside Rb1, Rg3 and Rf. While externally applied Rb1 and Rg3 had no effects on pacemaker potentials, Rf caused membrane depolarization. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker potentials and inhibited the Rf-induced membrane depolarization. Membrane depolarization induced by Rf was not inhibited by intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca 2+-free solution, thapsigargin, a Ca 2+-ATPase inhibitor of the endoplasmic reticulum, U-73122, a phospholipase C inhibitor, or 2-APB, an IP 3 receptor inhibitor, abolished the generation of pacemaker potentials and suppressed Rfinduced actions. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block Rf-induced effects on pacemaker potentials. These results suggest that ginsenoside Rf modulates the pacemaker activities of ICCs and therby regulates intestinal motility.
Molecules and Cells, 2011
The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract and transient... more The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract and transient receptor potential melastatin type 7 (TRPM7) is a candidate for pacemaker channels. The effect of the 5-lipoxygenase (5-LOX) inhibitors NDGA, AA861, MK886 and zileuton on pacemaking activity of ICCs was examined using the whole cell patch clamp technique. NDGA and AA861 decreased the amplitude of pacemaker potentials in ICC clusters, but the resting membrane potentials displayed little change, respectively. Also, perfusing NDGA and AA861 into the bath reduced both inward current and outward current in TRPM7like current in single ICC, respectively. But, they had no effects on Ca 2+ activated Clcurrents. The 5-LOX inhibitors MK886 and zileuton were, however, ineffective in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC, respectively. A specific TRPC3 inhibitor, pyrazole compound (Pyr3), and a specific TRPM4 inhibitor, 9-phenanthrol, had no effects in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC. These results suggest that, among the tested 5-LOX inhibitors, NDGA and AA861 modulate the pacemaker activities of the ICCs, and that the TRPM7 channel can affect intestinal motility.
The Korean Journal of Physiology and Pharmacology, 2010
Mast cells are activated by specific allergens and also by various nonspecific stimuli, which mig... more Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23∼ 25 o C) to a moderately high temperature (MHT, 37∼ 39 o C) to activate TRPV3/4, a high temperature (HT, 44∼ 46 o C) to activate TRPV1, or a very high temperature (VHT, 53∼ 55 o C) to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4αphorbol 12,13-didecanoate (4α PDD, 1μ M) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca 2+ ]c of HMC-1 cells was also increased by MHT or by 4α PDD. In summary, our present study indicates that HMC-1 cells express Ca 2+-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.
Planta medica, 2015
Cyperus rotundus is used as an analgesic and sedative in oriental medicine and has been reported ... more Cyperus rotundus is used as an analgesic and sedative in oriental medicine and has been reported to exhibit antinociceptive and anti-inflammatory effects. On the other hand, the transient receptor potential vanilloid 1 channel is a nonselective cation channel that senses various noxious chemical and thermal stimuli. However, it has recently been reported that the epidermally expressed transient receptor potential vanilloid 1 channel is involved in heat- and UV-induced skin aging. The aim of this study was to evaluate whether C. rotundus extract and its constituents can inhibit this channel. Ethylacetate and hexane fractions of the methanol extract were found to partially inhibit transient receptor potential vanilloid 1 channel activity, and at a concentration of 90 µM, oleanolic acid, which was one of three constituents isolated from the ethylacetate fraction, inhibited this activity by 61.4 ± 8.0 %. This is first electrophysiological study to be conducted on the effects of C. rotun...
PLoS ONE, 2013
Transthyretin (TTR) is a known carrier protein for thyroxine (T 4) and retinol-binding protein in... more Transthyretin (TTR) is a known carrier protein for thyroxine (T 4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTR kd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOG kd) did not have any effect on TTR gene expression. Both TTR kd and MYOG kd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T 4 uptake during myogenesis was observed in TTR kd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.
Pflugers Arch - Eur J Physiol, 2008
Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we f... more Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we found that WEHI-231, an immature B cell line, expresses voltage-independent K(+) channels called large-conductance background K( + ) channels (LK(bg)). Arachidonic acid (AA) has attracted attention because of its potential regulatory roles in the apoptosis of immature B cells. To elucidate the functional targets of AA, we investigated the effects of AA on membrane currents, voltages, and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) of WEHI-231 and Bal-17 cells that represent immature and mature mouse B cells, respectively. In whole-cell patch clamp, both Kv and IKCa1 were inhibited by AA. On the other hand, AA activated LK(bg) current and non-selective cationic (NSC) current in WEHI-231 while only NSC current in Bal-17. Inside-out patch clamp study showed that AA directly activates LK(bg). AA induced hyperpolarization of WEHI-231 and depolarization of Bal-17 cells, respectively. The selective functional expression of LK(bg) and their activation by AA were also confirmed in the immature B cells (B220(+)/AA4.1(+)) freshly isolated from mouse spleen. In fura-2 spectrofluorimetry, AA induced persistent increase in [Ca(2+)](c) of WEHI-231 cells, which was attenuated by KCl-induced depolarization. In Bal-17 cell, however, AA induced only a transient increase of [Ca(2+)](c). In summary, the novel type of background K(+) channels (LK(bg)) in immature B cells is strongly activated while the other K(+) channels (Kv and IKCa1) commonly expressed in lymphocytes are inhibited by AA. The hyperpolarization and augmentation of Ca(2+) influx by LK(bg) activation might play a role in the response of immature B cells to AA.
Cellular Physiology and Biochemistry, 2014
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCo... more This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2014
The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO2, 2... more The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO2, 2-5% P(O2)). However, functional studies of B cells in hypoxic conditions are sparse. Recently, we reported the expression in mouse B cells of TASK-2, a member of pH-sensitive two-pore domain K(+) channels with background activity. In this study, we investigated the response of K(+) channels to sustained PhyO2 (sustained hypoxia [SH], 3% P(O2) for 24 h) in WEHI-231 mouse B cells. SH induced voltage-independent background K(+) conductance (SH-K(bg)) and hyperpolarized the membrane potential. The pH sensitivity and the single-channel conductance of SH-K(bg) were consistent with those of TASK-2. Immunoblotting assay results showed that SH significantly increased plasma membrane expressions of TASK-2. Conversely, SH failed to induce any current following small interfering (si)TASK-2 transfection. Similar hypoxic upregulation of TASK-2 was also observed in splenic primary B cells. Mechanistica...
The Prostate, 2003
The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neuron... more The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.
The Journal of Physiology, 2007
In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C... more In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP 2) is not known yet. The mouse B cells express large conductance background K + channels (LK bg) that are inhibited by PIP 2. In inside-out patch clamp studies, the application of MgATP (1 mM) also inhibited LK bg due to the generation of PIP 2 by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK bg , which was antagonized by PIP 2 (> 1 μM) or higher concentration of MgATP (5 mM). The inhibition by PIP 2 was partially reversible. However, the application of methyl-β-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK bg activity and facilitated the activation by stretch. In cell-attached patches, LK bg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK bg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LK bg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCγ2 and PIP 2 hydrolysis of plasma membrane. The time dependence of PIP 2 hydrolysis and LK bg activation were similar. The presence of LK bg and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP 2 caused by stretch-activated PLC releases LK bg from the tonic inhibition by PIP 2 .
Proceedings of the National Academy of Sciences, 2013
Sequences meeting the above criteria were further classified by the Ribosomal Database Project (R... more Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.5 using training set 9 (46) from phylum to genus level." should instead appear as "Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.2 using training set 6 (46) from phylum to genus level."
Pflügers Archiv - European Journal of Physiology, 2006
Voltage-dependent, outwardly rectifying anion channels have been described in various cells inclu... more Voltage-dependent, outwardly rectifying anion channels have been described in various cells including lymphocytes. In this study, we found that murine B cells express the voltage-dependent slowly activating anion channels (VSACs). Using a whole-cell configuration, I (VSAC) in Bal-17 was induced by a sustained depolarization (>0 mV) which was remarkably facilitated at 35 degrees C (Q (10)=23 at 30 mV of clamp voltage). Substitution of extracellular Cl(-) with gluconate shifted the reversal potential to the right (35.7 mV). Gd(3+) (IC(50)=0.11 microM) significantly attenuated I (VSAC), but DIDS partially blocked I (VSAC). In addition, extracellular acidification suppressed I (VSAC) whereas alkalinization facilitated the channel activation. I (VSAC) was decreased by 90% at pH 6.35 and increased by 180% at pH 8.0. In cell-attached and inside-out patch clamps, depolarization slowly activated the anion channels of large conductance (approximately 270 pS) with multiple levels of subconductances. The single channel currents were also blocked by Gd(3+) and acidic pH. Furthermore, I (VSAC) was also observed in WEHI-231 (an immature B cell line) and freshly isolated splenic B cells of mice. In summary, murine B cells express unique voltage-dependent anion channels that show a strong sensitivity to both temperature and extracellular pH. Further investigation is required to understand the physiological roles of VSAC and its molecular identity.
Pflügers Archiv - European Journal of Physiology, 2008
K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells... more K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K+ channel (LK bg) in addition to the voltage-gated K+ channel (Kv) and Ca2+-activated K+ channel current (IKCa1). Mibefradil, a blocker of T-type Ca2+ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK bg current with half inhibitory concentration (IC50), 1.9 and 2.3 microM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca2+ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 microM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 microM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1.
Pfl�gers Archiv European Journal of Physiology, 2004
In this study, the short-circuit currents (I sc) of electrolyte absorption and secretion in neona... more In this study, the short-circuit currents (I sc) of electrolyte absorption and secretion in neonatal and adult rat colonic mucosa were compared and the role of Ca 2+ influx through luminal membranes examined in relation to the replenishment of intracellular Ca 2+ stores in colonic crypt cells. Neonatal tissues displayed higher amiloridesensitive I sc and larger increases of electrogenic Cl − secretion in response to an increase in cytosolic [Ca 2+ ] ([Ca 2+ ] c) or cAMP than found in adult colonic epithelium. Ca 2+-mediated Cl − secretion as reflected in the I sc responses to carbachol (I sc,CCh) showed milder "rundown" in neonates than in adult rats. We then employed the relatively stable I sc,CCh of the neonatal colon to investigate the polarity of Ca 2+ entry pathway after muscarinic stimulation. Repetitive stimulation with CCh under Ca 2+-free conditions emptied the intracellular Ca 2+ stores and abolished the I sc,CCh. Re-adding Ca 2+ to the basolateral perfusate rapidly restored I sc,CCh (about 71% of control in 10 min). In contrast, after re-adding Ca 2+ to the luminal perfusate only, the recovery of I sc,CCh took much longer and was incomplete, recovering to only 28% of control after 30 min. Recovery was accelerated by increasing [Ca 2+ ] in the luminal perfusate (5 mM) and blocked by the presence of Gd 3+ (100 µM) in the luminal perfusate. The above results suggest that, in addition to the predominant role of Ca 2+ entry through the basolateral membrane, the influx of Ca 2+ through luminal membranes might also play a role in the Ca 2+ homeostasis of colonic epithelial cells.
Molecules and Cells, 2013
Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transien... more Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca 2+ activated Clchannels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720-P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.
Molecules and Cells, 2010
Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) ar... more Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca(2+)] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca(2+)](c) in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca(2+) entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca(2+)](c) increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca(2+)](c) or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca(2+)](c). Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca(2+) was measured using Ba(2+) as a surrogate ion. Similarly, Ba(2+) influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca(2+)-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca(2+) release was insignificant in primary B cells and inWEHI-231.
Molecules and Cells, 2012
Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and p... more Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and pharmacological actions in various organs. However, little is known about the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker potentials by ginsenoside in the interstitial cells of Cajal (ICCs) using the whole-cell patch clamp technique in the current clamp mode. Among ginsenosides, we investigated the effects of ginsenoside Rb1, Rg3 and Rf. While externally applied Rb1 and Rg3 had no effects on pacemaker potentials, Rf caused membrane depolarization. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker potentials and inhibited the Rf-induced membrane depolarization. Membrane depolarization induced by Rf was not inhibited by intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca 2+-free solution, thapsigargin, a Ca 2+-ATPase inhibitor of the endoplasmic reticulum, U-73122, a phospholipase C inhibitor, or 2-APB, an IP 3 receptor inhibitor, abolished the generation of pacemaker potentials and suppressed Rfinduced actions. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block Rf-induced effects on pacemaker potentials. These results suggest that ginsenoside Rf modulates the pacemaker activities of ICCs and therby regulates intestinal motility.
Molecules and Cells, 2011
The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract and transient... more The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract and transient receptor potential melastatin type 7 (TRPM7) is a candidate for pacemaker channels. The effect of the 5-lipoxygenase (5-LOX) inhibitors NDGA, AA861, MK886 and zileuton on pacemaking activity of ICCs was examined using the whole cell patch clamp technique. NDGA and AA861 decreased the amplitude of pacemaker potentials in ICC clusters, but the resting membrane potentials displayed little change, respectively. Also, perfusing NDGA and AA861 into the bath reduced both inward current and outward current in TRPM7like current in single ICC, respectively. But, they had no effects on Ca 2+ activated Clcurrents. The 5-LOX inhibitors MK886 and zileuton were, however, ineffective in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC, respectively. A specific TRPC3 inhibitor, pyrazole compound (Pyr3), and a specific TRPM4 inhibitor, 9-phenanthrol, had no effects in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC. These results suggest that, among the tested 5-LOX inhibitors, NDGA and AA861 modulate the pacemaker activities of the ICCs, and that the TRPM7 channel can affect intestinal motility.
The Korean Journal of Physiology and Pharmacology, 2010
Mast cells are activated by specific allergens and also by various nonspecific stimuli, which mig... more Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23∼ 25 o C) to a moderately high temperature (MHT, 37∼ 39 o C) to activate TRPV3/4, a high temperature (HT, 44∼ 46 o C) to activate TRPV1, or a very high temperature (VHT, 53∼ 55 o C) to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4αphorbol 12,13-didecanoate (4α PDD, 1μ M) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca 2+ ]c of HMC-1 cells was also increased by MHT or by 4α PDD. In summary, our present study indicates that HMC-1 cells express Ca 2+-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.