Yangmi Kim

Functional expression of ion channels in mesenchymal stem cells derived from umbilical cord vein

Mesenchymal stem cells have the ability to renew and differentiate into various lineages of mesenchymal tissues. We used undifferentiated human mesenchymal‐like stem cells from human umbilical cord vein (hUC‐MSCs), a cell line which contains several mesenchymal cell markers. We characterized functional ion channels in cultured hUC‐MSCs with whole‐cell patch clamp and reverse transcription‐polymerase chain reaction (RT‐PCR). Three types of outward current were found in these cells: the Ca2+‐activated K+ channel (IKCa), a transient outward K+ current (Ito), and a delayed rectifier K+ current (IKDR). IKCa and IKDR were totally suppressed by tetraethylammonium, and IKCa was sensitive to a specific blocker, iberiotoxin. Ito was inhibited by 4‐aminopyridine. Another type of inward rectifier K+ current (Kir) was also detected in approximately 5% of hUC‐MSCs. Elevation of external potassium ion concentration increased the Kir current amplitude and positively shifted its reversal potential. In addition, inward Na+ current (INa) was found in these cells (∼30%); the current was blocked by tetrodotoxin and verapamil. In the RT‐PCR analysis, Kv1.1, Kv4.2, Kv1.4, Kir2.1, heag1, MaxiK, hNE‐Na, and TWIK‐1 were detected. These results suggested that multiple functional ion channel currents, IKCa, IKDR, Ito, INa, and Kir, are expressed in hUC‐MSCs.

Acetylcholine-induced phosphatidylinositol 4,5-bisphosphate depletion does not cause short-term desensitization of G protein-gated inwardly rectifying K+ current in mouse atrial myocytes.

Depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by phenylephrine or endothelin causes the inhibition of acetylcholine-activated K+ current (IKACh) in atrial myocytes. In the present study, we have investigated the hypothesis that muscarinic receptor induced PIP2 depletion also causes inhibition of IKACh, resulting in desensitization. We confirmed the expression of Gq-coupled muscarinic receptors in mouse atrial myocytes using reverse transcriptase-polymerase chain reaction. The involvement of M1 and M3receptors in desensitization is examined using specific antagonists, 4-DAMP and pirenzepine, but they significantly reduced peak IKACh, implying nonspecific M2 blockade. When ACh-induced phosphoinositide depletion was specifically inhibited using PLCβ1 knock-out mice, the extent of desensitization during 4 min was 47.5 ± 3.2%, which was not different from that in wild type (46.8 ± 2.1%). Phenylephrine-induced phosphoinositide hydrolysis and phenylephrine-induced inhibition of IKACh were not affected by PLCβ1 knock-out. To facilitate PIP2depletion, replenishment of PIP2 was blocked by wortmannin. Wortmannin did not affect the desensitization and the recovery from desensitization. These results suggest that PIP2 depletion by acetylcholine does not contribute to short-term desensitization of IKACh. The differential regulation of IKACh by different phospholipase C-linked receptors may imply that receptor co-localization is required for PIP2 to act as a signaling molecule.

High expression of large-conductance Ca2+-activated K+ channel in the CD133+ subpopulation of SH-SY5Y neuroblastoma cells

Solid tumors contain a population of cancer stem cells (CSCs), and CD133 is widely used as a CSCs marker. We investigated the differences between CD133(+) and CD133(-) cells from the neuroblastoma cell line SH-SY5Y in terms of the expressions of voltage-gated ion channels. A CD133(+) enriched (>60%) population was isolated, and a subsequent whole-cell voltage-clamp study showed that these cells predominantly express TEA-sensitive outward K(+) currents (I(K,TEA)) and TTX-sensitive voltage-gated inward Na(+) currents (I(Na)). Cell-attached single channel recordings demonstrated higher density of large-conductance (155pS) channel in CD133(+) cells than in CD133(-) cells. The TEA-sensitivity and single channel conductance indicated the large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). Furthermore, RT-PCR analysis of 22 transcripts of voltage-gated ion channels in SH-SY5Y cells showed the expressions of Cav1.3, Kir2.1, Kv1.4, Kv2.1, Kv4.2, Kv7.1, BK(Ca), and Nav1.7, and those of BK(Ca) and Nav1.7 were higher in CD133(+) than in CD133(-) cells. In addition, the increase of cytosolic Ca(2+) concentration ([Ca(2+)](c)) in response to ionomycin (a Ca(2+) ionophore) was higher and more sustained in CD133(+) than in CD133(-) cells. Plausibly membrane hyperpolarization via BK(Ca) might be responsible for the augmented Ca(2+) influx observed in CD133(+) cells. The physiological implications of the differential expression of BK(Ca) and Nav1.7 in CSCs require further investigation.

HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells

The effects of histone deacetylase (HDAC) inhibitors on epithelial-mesenchymal transition (EMT) differ in various types of cancers. We investigated the EMT phenotype in four colon cancer cell lines when challenged with HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) with or without transforming growth factor-β1 (TGF-β1) treatment. Four colon cancer cell lines with different phenotypes in regards to tumorigenicity, microsatellite stability and DNA mutation were used. EMT phenotypes were assessed by the expression of E-cadherin and vimentin using western blot analysis, immunofluorescence, quantitative real-time RT-PCR following treatment with TSA (100 or 200 nM) or VPA (0.5 mM) with or without TGF-β1 (5 ng/ml) for 24 h. Biological EMT phenotypes were also evaluated by cell morphology, migration and invasion assays. TSA or VPA induced mesenchymal features in the colon carcinoma cells by a decrease in E-cadherin and an increase in vimentin expression at the mRNA and protein levels. Confocal microscopy revealed membranous attenuation or nuclear translocation of E-cadherin and enhanced expression of vimentin. These responses occurred after 6 h and increased until 24 h. Colon cancer cells changed from a round or rectangular shape to a spindle shape with increased migration and invasion ability following TSA or VPA treatment. The susceptibility to EMT changes induced by TSA or VPA was comparable in microsatellite stable (SW480 and HT29) and microsatellite unstable cells (DLD1 and HCT116). TSA or VPA induced a mesenchymal phenotype in the colon carcinoma cells and these effects were augmented in the presence of TGF-β1. HDAC inhibitors require careful caution before their application as new anticancer drugs for colon cancers.

Diversity of Ion Channels in Human Bone Marrow Mesenchymal Stem Cells from Amyotrophic Lateral Sclerosis Patients

Human bone marrow mesenchymal stem cells (hBM-MSCs) represent a potentially valuable cell type for clinical therapeutic applications. The present study was designed to evaluate the effect of long-term culturing (up to 10(th) passages) of hBM-MSCs from eight individual amyotrophic lateral sclerosis (ALS) patients, focusing on functional ion channels. All hBM-MSCs contain several MSCs markers with no significant differences, whereas the distribution of functional ion channels was shown to be different between cells. Four types of K(+) currents, including noise-like Ca(+2)-activated K(+) current (IK(Ca)), a transient outward K(+) current (I(to)), a delayed rectifier K(+) current (IK(DR)), and an inward-rectifier K(+) current (K(ir)) were heterogeneously present in these cells, and a TTX-sensitive Na(+) current (I(Na,TTX)) was also recorded. In the RT-PCR analysis, Kv1.1, heag1, Kv4.2, Kir2.1, MaxiK, and hNE-Na were detected. In particular, I(Na,TTX) showed a significant passage-dependent increase. This is the first report showing that functional ion channel profiling depend on the cellular passage of hBM-MSCs.

The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from -19.9 mV±0.8 (n=116) to -8.5 mV±1.4 (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.

Statin pretreatment inhibits the lipopolysaccharide-induced epithelial-mesenchymal transition via the downregulation of toll-like receptor 4 and nuclear factor-κB in human biliary epithelial cells.

Epithelial‐mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays an important role in biliary fibrosis. This study investigated the effects of simvastatin on the lipopolysaccharide (LPS)‐induced EMT and related signal pathways in BECs.

Statin pretreatment inhibits the LPS-induced EMT via the downregulation of TLR4 and NF-κB in human biliary epithelial cells.

Epithelial‐mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays an important role in biliary fibrosis. This study investigated the effects of simvastatin on the lipopolysaccharide (LPS)‐induced EMT and related signal pathways in BECs.

Functional expression of TREK1 channel in human bone marrow and human umbilical cord vein-derived mesenchymal stem cells

Abstract Human bone marrow or human umbilical cord vein derived-mesenchymal stem cells (hBM-MSCs or hUC-MSCs) have known as a potentially useful cell type for clinical therapeutic applications. We investigated two-pore domain potassium (K2P) channels in these cells. K2P channels play a major role in setting the resting membrane potential in many cell types. Among them, TREK1 is targets of hydrogen, hypoxia, polyunsaturated fatty acids, antidepressant, and neurotransmitters. We investigated whether hBM-MSCs and hUC-MSCs express functionalTREK1 channel using RT-PCR analysis and patch clamp technique. Potassium channel with a single channel conductance of 100 pS was found in hUC-MSCs and BM-MSCs and the channel was activated by membrane stretch (-5 mmHg ~ -15 mmHg), arachidonic acid (10 μM) and intracellular acidosis (pH 6.0). These electrophysiologicalproperties were similar to those of TREK1. Our results suggest that TREK1 is functionally present in hBM-MSCsand hUC-MSCs, where they contribute to its resting membrane potential.

Comparative analysis of blood glucose test results on the forearm, finger, and vein

Abstract Capillary blood sampling on the forearm reduces pain caused by skin puncture. The present study comparedthe blood glucose test results performed at different sampling sites of the forearm, finger, and vein to evaluate clinicalvalidity of this alternative site blood sampling technique. Subjects numbered 555 including 61 diabetic patients participated to measure the glucose concentration on the finger (G F ) and the forearm (G A ) with a portable glucometerunder overnight fasting state. Then, the venous glucose concentration (G V ) was measured in 514 subjects in less than1 hour. The test results were analyzed by simple linear regression, intraclass correlation, and Passing-Bablok regression techniques. G A was highly correlated with G F or G V showing the correlation coefficients (r) of approximately 0.97 (P<0.0001) in the normal group. The patient group also resulted similarly high correlation with only slightly lower r value. The mean differences in glucose concentration were less than ±10mg/dL regardless ofthe sampling sites. Intraclass correlation coefficients were slightly smaller than r but very much similar in value inboth groups. The 95% confidence intervals of the slope as well as the intercept in the Passing-Bablok regressionanalysis were <±20% and <±20mg/dL, respectively, which were within the clinically acceptable ranges. These threestatistical techniques introduced in the present study well demonstrated the consistency of G