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ABSTRACT: BACKGROUND AND PURPOSE: The intermediate-conductance Ca(2+) -activated K(+) channel KCa 3.1 modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of KCa 3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. EXPERIMENTAL APPROACH: The expression patterns of KCa 3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized but non-challenged (Ox+/-), and Ox-sensitized and -challenged (Ox+/+)] using real-time PCR, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of KCa 3.1 blockade were examined by the administration of selective KCa 3.1 blockers. KEY RESULTS: Significant up-regulation of KCa 3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, KCa 3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor, REST was significantly down-regulated. Pharmacological blockade of KCa 3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0 /G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the KCa 3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. CONCLUSIONS AND IMPLICATIONS: The up-regulation of KCa 3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice, and KCa 3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.
British Journal of Pharmacology 04/2013; · 4.41 Impact Factor
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ABSTRACT: The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counter-ion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate (IP3) receptors produces global Ca(2+) transients causing contraction. Tric-a-knockout mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of an SMC-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell-surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired IP3-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice, together with Tric-a-knockout mice, indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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ABSTRACT: Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca(2+)-activated K(+) channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1-ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.
Biochemical and Biophysical Research Communications 12/2012; · 2.48 Impact Factor
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ABSTRACT: In smooth muscles (SMs), cytosolic Ca(2+) ([Ca(2+)](cyt)) dynamics during an action potential are triggered by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) in the plasma membrane. The physiological significance of Ca(2+) amplification by subsequent Ca(2+) release through ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR) is still a matter of topics in SMs. In the present study, depolarization-evoked local Ca(2+) dynamics in Ca(2+) microdomain were imaged using total internal reflection fluorescence (TIRF) microscopy in mouse urinary bladder SM cells (UBSMCs). Upon depolarization under whole-cell voltage-clamp, the rapid and local elevation of [Ca(2+)](cyt) was followed by larger [Ca(2+)](cyt) increase with propagation occurred in a limited TIRF zone within ∼200nm from cell surface. The depolarization-evoked [Ca(2+)](cyt) increase in a TIRF zone was abolished or greatly reduced by the pretreatment with Cd(2+) or ryanodine, respectively. The initial local [Ca(2+)](cyt) increases were mediated by Ca(2+) influx through single or clustered VDCCs as Ca(2+) sparklets, and the following step was elicited by Ca(2+)-induced Ca(2+) release (CICR) through RyR from SR. The depolarization-induced outward currents, mainly due to large-conductance Ca(2+)-activated K(+) channel activation, were also markedly reduced by Cd(2+) and ryanodine. In addition, TIRF analyses showed that the fluorescent signals of individual or clustered VDCC distributed in relatively uniform fashion and that a subset of RyRs in the subplasmalemmal SR also located in TIRF zone. In conclusion, fast TIRF imaging successfully demonstrated two step Ca(2+) events upon depolarization in Ca(2+) microdomain of UBSMCs; the initial Ca(2+) influx as Ca(2+) sparklets through discrete VDCC or their clusters and the following CICR via the activation of loosely coupled RyRs in SR located in the Ca(2+) microdomains.
Biochemical and Biophysical Research Communications 09/2012; 427(1):54-9. · 2.48 Impact Factor
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Manabu Murakami,
Hisao Yamamura,
Agnieszka Murakami,
Tadashi Okamura,
Kazuo Nunoki,
Minori Mitui-Saito,
Katsuhiko Muraki,
Takuzou Hano, Yuji Imaizumi,
Veit Flockerzi,
Teruyuki Yanagisawa
Journal of Cardiovascular Pharmacology 08/2012; 36:S69-S73. · 2.29 Impact Factor
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ABSTRACT: The involvement of inositol 1,4,5-trisphosphate (IP(3)) formation in the voltage-dependent regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in smooth muscle cells of the porcine coronary artery. Slow ramp depolarization from -90 to 0 mV induced progressive [Ca(2+)](i) increase. The slope was reduced or increased in the presence of Cd(2+) or (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxlic acid methyl ester (Bay K 8644), respectively. The decrease in [Ca(2+)](i) via the membrane hyperpolarization induced by K(+) channel openers (levcromakalim and Evans blue) under current clamp was identical to that under voltage clamp. The step hyperpolarization from -40 to -80 mV reduced [Ca(2+)](i) uniformly over the whole-cell area with a time constant of ∼10 s. The [Ca(2+)](i) at either potential was unaffected by heparin, an inhibitor of IP(3) receptors. Alternatively, [Ca(2+)](i) rapidly increased in the peripheral regions by depolarization from -80 to 0 mV and stayed at that level (∼400 nM) during a 60-s pulse. When the pipette solution contained IP(3) pathway blockers [heparin, 2-aminoethoxydiphenylborate, xestospongin C, or 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], the peak [Ca(2+)](i) was unchanged, but the sustained [Ca(2+)](i) was gradually reduced by ∼250 nM within 30 s. In the presence of Cd(2+), a long depolarization period slightly increased the [Ca(2+)](i), which was lower than that in the presence of heparin alone. In coronary arterial myocytes, the sustained increase in the [Ca(2+)](i) during depolarization was partly caused by the Ca(2+) release mediated by the enhanced formation of IP(3). The initial [Ca(2+)](i) elevation triggered by the Ca(2+) influx though voltage-dependent Ca(2+) channels may be predominantly responsible for the activation of phospholipase C for IP(3) formation.
Journal of Pharmacology and Experimental Therapeutics 05/2012; 342(2):486-96. · 3.83 Impact Factor
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ABSTRACT: To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.
Journal of Biomolecular Screening 04/2012; 17(6):773-84. · 2.05 Impact Factor
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ABSTRACT: The large-conductance Ca(2+)-activated K(+) (K(Ca)1.1, BK) channel has pivotal roles in the regulation of vascular tone. To clarify the molecular dynamics of BK channels and their functionally coupled protein on the membrane surface, we examined single-molecule imaging of fluorescent-labeled BK subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. The dynamic mobility of yellow fluorescent protein (YFP)-tagged BKα subunit (BKα-YFP) expressed in human embryo kidney 293 (HEK) cells was detected in TIRF regions at the level of individual channels and their clusters on the plasma membrane with a diffusion coefficient of 6.7 × 10(3) nm(2)/s. When BKα-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BKβ1 subunit (BKβ1-CFP) in HEK cells, the mobility was reduced by ∼50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BKα subunit (BKα-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BKα subunits, rather than homotetrameric BKα-GFP. Movement of BKα-YFP in VSMCs (0.29 × 10(3) nm(2)/s) was far more restricted than BKα-YFP/BKβ1-CFP in HEK cells (2.5 × 10(3) nm(2)/s). Actin disruption by pretreatment with cytochalasin D in VSMCs appeared to increase the mobile behavior of BKα-YFP, which was then significantly reduced by addition of jasplakinolide. Most BKα-YFP colocalized with caveolin 1 (Cav1)-CFP in VSMCs, but unexpectedly not frequently in HEK cells. Fluorescence resonance energy transfer analyses showed the direct interaction between BKα-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BKα molecules on the membrane surface are strongly restricted or regulated by its auxiliary β-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs.
AJP Cell Physiology 02/2012; 302(8):C1257-68. · 3.54 Impact Factor
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ABSTRACT: Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel encoded by K(Ca)1.1 plays an important role in the control of smooth muscle tone by modulating membrane potential and intracellular Ca(2+) mobilization. BK(Ca) channel is functionally expressed in prostatic smooth muscle cells, and is activated by α(1)-adrenoceptor agonists. The main objective of this study was to elucidate the pathophysiological significance of changes in prostatic K(Ca)1.1 expressions in benign prostatic hyperplasia (BPH). Our previous study has shown that K(Ca)3.1 encoding intermediate-conductance K(Ca) (IK(Ca)) channel is up-regulated in stromal cells of implanted urogenital sinuses (UGSs) of stromal hyperplasia BPH model rats and in those of prostatic tissues from BPH patients. In the present study, the results from real-time polymerase chain reaction (PCR), Western blot, and immunohistochemical analyses showed significant down-regulation of K(Ca)1.1 transcripts and proteins and negative correlation between K(Ca)1.1 and K(Ca)3.1 transcript expressions in prostatic stromal cells of both BPH model rats and BPH patients. Corresponding to down-regulation of K(Ca)1.1 expression in stromal cells of implanted UGSs, membrane depolarization by application of the BK(Ca) channel blocker was disappeared. Down-regulation of K(Ca)1.1 may be involved in the phenotype switch from contractile profile to proliferative one in prostatic stromal cells of BPH patients.
Biological & Pharmaceutical Bulletin 01/2012; 35(5):737-44. · 1.66 Impact Factor
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Daiju Yamazaki,
Yasuharu Tabara,
Satomi Kita,
Hironori Hanada,
Shinji Komazaki,
Daisuke Naitou,
Aya Mishima,
Miyuki Nishi,
Hisao Yamamura,
Shinichiro Yamamoto, [......],
Kei Kamide,
Toshio Ogihara,
Akira Hata,
Satoshi Umemura,
Masayoshi Soma,
Norio Takahashi, Yuji Imaizumi,
Tetsuro Miki,
Takahiro Iwamoto,
Hiroshi Takeshima
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ABSTRACT: TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.
Cell metabolism 08/2011; 14(2):231-41. · 17.35 Impact Factor
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ABSTRACT: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K(+) channel (K(ir)2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K(ir) channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca(2+) concentration due to Ca(2+) influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K(ir)2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.
Biochemical and Biophysical Research Communications 07/2011; 411(2):293-8. · 2.48 Impact Factor
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ABSTRACT: Recently, a new experimental stromal hyperplasia animal model corresponding to clinical benign prostatic hyperplasia (BPH) was established. The main objective of this study was to elucidate the roles of the intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) in the implanted urogenital sinus (UGS) of stromal hyperplasia BPH model rats. Using DNA microarray, real-time polymerase chain reaction, Western blot, and/or immunohistochemical analyses, we identified the expression of K(Ca)3.1 and its transcriptional regulators in implanted UGS of BPH model rats and prostate needle-biopsy samples and surgical prostate specimens of BPH patients. We also examined the in vivo effects of a K(Ca)3.1 blocker, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), on the proliferation index of implanted UGS by measurement of UGS weights and proliferating cell nuclear antigen immunostaining. K(Ca)3.1 genes and proteins were highly expressed in implanted UGS rather than in the normal host prostate. In the implanted UGS, the gene expressions of two transcriptional regulators of K(Ca)3.1, repressor element 1-silencing transcription factor and c-Jun, were significantly down- and up-regulated, and the regulations were correlated negatively or positively with K(Ca)3.1 expression, respectively. Positive signals of K(Ca)3.1 proteins were detected exclusively in stromal cells, whereas they were scarcely immunolocalized to basal cells of the epithelium in implanted UGS. In vivo treatment with TRAM-34 significantly suppressed the increase in implanted UGS weights compared with the decrease in stromal cell components. Moreover, significant levels of K(Ca)3.1 expression were observed in human BPH samples. K(Ca)3.1 blockers may be a novel treatment option for patients suffering from BPH.
Journal of Pharmacology and Experimental Therapeutics 05/2011; 338(2):528-36. · 3.83 Impact Factor
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ABSTRACT: The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.
Journal of Biological Chemistry 02/2011; 286(19):16940-52. · 4.77 Impact Factor
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Folia Pharmacologica Japonica 01/2011; 138(6):229-33.
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ABSTRACT: Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca(2+) and electric activities in ICC were performed by employing fluorescent Ca(2+) imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca(2+) antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca(2+) oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT(3) receptor antagonist suppressed spontaneous Ca(2+) activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT(3) receptor agonist, restored it. GR113808, a selective antagonist for 5-HT(4), and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT(3) receptors, had little effect on ICC Ca(2+) activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT(3) receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT(3) receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.
PLoS ONE 01/2011; 6(9):e24928. · 4.09 Impact Factor
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ABSTRACT: Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca²(+) concentration ([Ca²(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca²(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca²(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ∼15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba²(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba²(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba²(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca²(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.
AJP Cell Physiology 10/2010; 300(1):C75-86. · 3.54 Impact Factor
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ABSTRACT: In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca(2+)-activated K(+) (K(Ca)) channels in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca(2+)](i) rise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H(1) receptors. The histamine-induced membrane hyperpolarization was attenuated to approximately 50% by large-conductance K(Ca) (BK) channel blockers, and further reduced by intermediate (IK) and small conductance K(Ca) (SK) channel blockers. The tonic component of histamine-induced [Ca(2+)](i) rise strongly depended on the presence of extracellular Ca(2+) ([Ca(2+)](o)) and was markedly reduced by La(3+) or Gd(3+) but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The K(Ca) blocker cocktail also significantly reduced the store-operated Ca(2+) entry (SOCE), which was induced by Ca(2+) addition after store-depletion by thapsigargin in [Ca(2+)](o) free solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to K(Ca) channel activation contributes to sustained Ca(2+) entry mainly through SOCE channels in OUMS-27 cells. Thus, K(Ca) channels appear to play an important role in the positive feedback mechanism of [Ca(2+)](i) regulation in chondrocytes in the presence of articular cartilage inflammation.
AJP Cell Physiology 04/2010; 298(4):C786-97. · 3.54 Impact Factor
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ABSTRACT: The rise of Ca(2+) concentration ([Ca(2+)](i)) by reducing external Na(+) in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na(+)/Ca(2+) exchanger type-1.3 (NCX1.3(tg/tg)) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3(tg/tg). The Ca(2+) release by caffeine in UBSMCs was comparable between NCX1.3(tg/tg) and WT, but [Ca(2+)](i) decay was faster in NCX1.3(tg/tg). Contractions induced by acetylcholine, 60 mM K(+), or electrical stimulation were significantly smaller in UB segments of NCX1.3(tg/tg). NCX worked in Ca(2+)-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3(tg/tg).
Journal of Pharmacological Sciences 02/2010; 112(3):373-7. · 2.08 Impact Factor
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ABSTRACT: The contribution of Cl(-) conductance relative to that of K(+) in the regulation of membrane potential was examined using OUMS-27 cells, a model cell-line of human chondrocytes. Application of 100 microM niflumic acid (NFA) and other anion-channel blockers induced significant membrane hyperpolarization. The NFA-sensitive membrane current under voltage-clamp was predominantly Cl(-) current. Application of NFA induced small but significant increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and markedly enhanced the late component of [Ca(2+)](i) rise induced by 1 microM histamine. In conclusion, Cl(-) conductance substantially contributes to the regulation of resting membrane potential and [Ca(2+)](i) in OUMS-27 cells.
Journal of Pharmacological Sciences 01/2010; 113(1):94-9. · 2.08 Impact Factor
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ABSTRACT: Large-conductance Ca(2+)-activated K(+ )(BK) channel regulates action potential firing in pancreatic β-cells. We cloned novel spliced variants of the BK-channel β(2)-subunit (BKβ2b), which consisted of 36 amino acids including the N-terminal in the original human BKβ2 (BKβ2a), from human and rodent pancreas. Real-time PCR analysis showed the abundant expression of BKβ2b transcripts in human and rodent pancreas and also in the RINm5f insulinoma cell line. In addition, up-regulation of both BK-channel α-subunit (BKα) and BKβ2b transcripts was observed in pancreas tissues from diabetes mellitus patients. In HEK293 cells co-expressing BKα and BKβ2b, the inactivation of BK-channel currents, which is typical for BKα + BKβ2a, was not observed, and electrophysiological and pharmacological properties of BKα + BKβ2b were almost identical to those of BKα alone. In HEK293 cells stably expressing BKα, the transient co-expression of yellow fluorescence protein (YFP)-tagged BKβ2a proteins resulted in their distribution along the cell membrane. In contrast, the co-expression of YFP-tagged BKβ2b with BKα showed diffusely distributed fluorescence signals throughout the cell body. Taken together, the predominant splicing of BKβ2b versus that of BKβ2a presumably enhances the contribution of BK channels to membrane potential and may possibly be a factor modulating insulin secretion in a suppressive manner in pancreatic β-cells.
Journal of Pharmacological Sciences 01/2010; 114(2):198-205. · 2.08 Impact Factor
