24 results on '"Dongki Yang"'
Search Results
2. Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis
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Dongki Yang, Seon-Hee Kim, and Young-An Bae
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Flatworms ,RC955-962 ,Gene Expression ,Eggshell formation ,Biochemistry ,Arctic medicine. Tropical medicine ,Gene expression ,Bile ,Parasite hosting ,Hypoxia ,Phylogeny ,Zebrafish ,Clonorchis ,Clonorchis sinensis ,Myoglobin ,Eukaryota ,Neurochemistry ,Globins ,Cell biology ,Chemistry ,Infectious Diseases ,Nitrosative Stress ,Physical Sciences ,Trematoda ,Neurochemicals ,Public aspects of medicine ,RA1-1270 ,Oxidation-Reduction ,Research Article ,Chemical Elements ,Biology ,Nitric Oxide ,Trematodes ,Immune system ,Helminths ,Genetics ,Animals ,Humans ,Metacercariae ,Globin ,Gene ,Nitrites ,Nitrates ,Host (biology) ,Organisms ,Chemical Compounds ,Public Health, Environmental and Occupational Health ,Biology and Life Sciences ,Proteins ,Cell Biology ,biology.organism_classification ,Invertebrates ,Oxygen ,Carcinogens ,Zoology ,Neuroscience - Abstract
Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity., Author summary Trematode parasites that invade mammalian tissues have long been believed to produce bioenergy via anaerobic respiration in their definitive hosts. However, recent studies have revealed that these parasites require considerable amounts of oxygen for the generation of hard eggshells during sexual reproduction as well as energy metabolism. Despite these findings, information on the biological mechanisms and relevant molecules responsible for oxygen uptake in the host environment remains largely elusive. Clonorchis sinensis is a carcinogenic trematode parasite that causes clonorchiasis in humans by infecting the bile ducts. Here, we investigated globin genes/proteins in the liver fluke. The genome of C. sinensis encoded at least five globin paralogs (CsMb1, CsMb2, CsMb3, CsNgb, and CsGbX). Temporal expression of these globin genes coincided with the sexual maturation of C. sinensis. Based on the histological localities and induction profiles upon hypoxia, it could be postulated that the oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms by CsMb1 and CsMb3, respectively, for energy metabolism and eggshell formation. Other globin homologs were likely to perform non-respiratory functions. In addition, the oxygenated form of secreted CsMb1 seemed to participate in the scavenging of nitric oxide generated by host immune cells via its nitric oxide dioxygenase activity to increase the survival of the parasite.
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- 2021
3. Impaired phagocytosis of apoptotic cells causes accumulation of bone marrow-derived macrophages in aged mice
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Yu-Hoi Kang, Dae Ho Lee, Hyo-Jung Kim, Jin Ku Kang, Gi Jeong Cheon, Ok-Hee Kim, Dongki Yang, Byung-Chul Oh, and Sang Chul Park
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Male ,0301 basic medicine ,Aging ,Phagocytosis ,CD14 ,Anti-Inflammatory Agents ,Lipopolysaccharide Receptors ,Apoptosis ,Bone Marrow Cells ,Inflammation ,Biochemistry ,Jurkat cells ,Jurkat Cells ,Mice ,03 medical and health sciences ,medicine ,Animals ,Humans ,Macrophage ,Molecular Biology ,Chemistry ,Macrophages ,Cell Differentiation ,Articles ,General Medicine ,Interleukin-10 ,Mice, Inbred C57BL ,Interleukin 10 ,030104 developmental biology ,medicine.anatomical_structure ,IL-10 ,Cancer research ,Cytokines ,Bone marrow ,medicine.symptom - Abstract
Accumulation of tissue macrophages is a significant characteristic of disease-associated chronic inflammation, and facilitates the progression of disease pathology. However, the functional roles of these bone marrow-derived macrophages (BMDMs) in aging are unclear. Here, we identified agedependent macrophage accumulation in the bone marrow, showing that aging significantly increases the number of M1 macrophages and impairs polarization of BMDMs. We found that age-related dysregulation of BMDMs is associated with abnormal overexpression of the anti-inflammatory interleukin-10. BMDM dysregulation in aging impairs the expression levels of pro-inflammatory cytokines and genes involved in B-cell maturation and activation. Phagocytosis of apoptotic Jurkat cells by BMDMs was reduced because of low expression of phagocytic receptor CD14, indicating that increased apoptotic cells may result from defective phagocytosis of apoptotic cells in the BM of aged mice. Therefore, CD14 may represent a promising target for preventing BMDM dysregulation, and macrophage accumulation may provide diagnostic and therapeutic clues. [BMB Reports 2017; 50(1): 43-48].
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- 2017
4. Beyond Lipid Signaling: Pleiotropic Effects of Diacylglycerol Kinases in Cellular Signaling
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Jaehong Kim, Dongki Yang, and Jae Ang Sim
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Cell signaling ,diacylglycerol kinase ,Phosphatidic Acids ,Review ,Catalysis ,Diglycerides ,lcsh:Chemistry ,Inorganic Chemistry ,chemistry.chemical_compound ,Neoplasms ,Diabetes Mellitus ,Animals ,Humans ,Protein Isoforms ,tumor microenvironment ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,Diacylglycerol kinase ,Inflammation ,chemistry.chemical_classification ,Tumor microenvironment ,diacylglycerol ,Kinase ,Organic Chemistry ,General Medicine ,Lipid signaling ,Phosphatidic acid ,tissue microenvironment ,Computer Science Applications ,Cell biology ,phosphatidic acid ,Enzyme ,lcsh:Biology (General) ,lcsh:QD1-999 ,chemistry ,lipid signaling ,lipids (amino acids, peptides, and proteins) ,Nervous System Diseases ,Signal Transduction - Abstract
The diacylglycerol kinase family, which can attenuate diacylglycerol signaling and activate phosphatidic acid signaling, regulates various signaling transductions in the mammalian cells. Studies on the regulation of diacylglycerol and phosphatidic acid levels by various enzymes, the identification and characterization of various diacylglycerol and phosphatidic acid-regulated proteins, and the overlap of different diacylglycerol and phosphatidic acid metabolic and signaling processes have revealed the complex and non-redundant roles of diacylglycerol kinases in regulating multiple biochemical and biological networks. In this review article, we summarized recent progress in the complex and non-redundant roles of diacylglycerol kinases, which is expected to aid in restoring dysregulated biochemical and biological networks in various pathological conditions at the bed side.
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- 2020
5. STIM-TRP Pathways and Microdomain Organization: Ca
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Dora, Bodnar, Woo Young, Chung, Dongki, Yang, Jeong Hee, Hong, Archana, Jha, and Shmuel, Muallem
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Membrane Microdomains ,Cell Membrane ,Animals ,Humans ,Membrane Proteins ,Calcium ,Stromal Interaction Molecule 1 ,Endoplasmic Reticulum ,Calcium Release Activated Calcium Channels ,TRPC Cation Channels - Abstract
Ca
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- 2017
6. Mitochondrial Retrograde Signalling and Metabolic Alterations in the Tumour Microenvironment
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Dongki Yang and Jaehong Kim
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Cell Nucleus ,retrograde signalling ,Metabolic reprogramming ,EMT ,Cancer ,Review ,General Medicine ,Mitochondrion ,Biology ,medicine.disease ,Models, Biological ,Mitochondria ,Cell biology ,lcsh:Biology (General) ,Metabolome ,Tumor Microenvironment ,medicine ,Retrograde signaling ,Animals ,Humans ,metabolic reprogramming ,tumour microenvironment ,lcsh:QH301-705.5 ,Signal Transduction - Abstract
This review explores the molecular mechanisms that may be responsible for mitochondrial retrograde signalling related metabolic reprogramming in cancer and host cells in the tumour microenvironment and provides a summary of recent updates with regard to the functional modulation of diverse cells in the tumour microenvironment.
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- 2019
7. Activation of TRPC4β by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron
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Ju Hong Jeon, Jinhong Wie, Hana Kim, Dongki Yang, Hyun-Jin Kim, In Gyu Kim, Insuk So, Seung Eon Roh, Nam Hyuk Cho, Jinsung Kim, Jaepyo Jeon, David E. Kang, and Kyu Pil Lee
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Cell Membrane Permeability ,Neurite ,Physiology ,Neurogenesis ,Gi alpha subunit ,Biology ,Hippocampal formation ,TRPC5 ,Hippocampus ,TRPC4 ,Mice ,Transient receptor potential channel ,GTP-Binding Proteins ,Ca2+/calmodulin-dependent protein kinase ,Neurites ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,TRPC ,TRPC Cation Channels ,Dendrites ,Cell Biology ,Cations, Monovalent ,Receptors, Muscarinic ,Cell biology ,Mice, Inbred C57BL ,HEK293 Cells ,Biochemistry ,Guanosine 5'-O-(3-Thiotriphosphate) ,Calcium ,GTP-Binding Protein alpha Subunit, Gi2 ,Calcium-Calmodulin-Dependent Protein Kinase Type 2 ,Ion Channel Gating ,Porosity ,Signal Transduction - Abstract
The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4β and the TRPC4β-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.
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- 2013
8. EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage
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Hye Seung Jung, Eun Young Park, Eung Hwi Kim, Dongki Yang, Hee-Sook Jun, and Mi Hwi Kim
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0301 basic medicine ,medicine.medical_specialty ,NF-E2-Related Factor 2 ,Biology ,Toxicology ,medicine.disease_cause ,Antioxidants ,03 medical and health sciences ,chemistry.chemical_compound ,Islets of Langerhans ,Internal medicine ,Cell Line, Tumor ,medicine ,Animals ,LY294002 ,Protein kinase A ,PI3K/AKT/mTOR pathway ,Pharmacology ,chemistry.chemical_classification ,Reactive oxygen species ,Venoms ,Glutathione ,Cell biology ,Rats ,Enzyme Activation ,Oxidative Stress ,Protein Kinase C-delta ,030104 developmental biology ,Endocrinology ,chemistry ,Exenatide ,Beta cell ,Peptides ,Rottlerin ,Oxidative stress - Abstract
Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H2O2. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress.
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- 2016
9. The Solute Carrier 26 Family of Proteins in Epithelial Ion Transport
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Shmuel Muallem, Dongki Yang, Nikolay Shcheynikov, and Michael R. Dorwart
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Protein Conformation ,Physiology ,Anion Transport Proteins ,Molecular Sequence Data ,Epithelial Cells ,Biology ,medicine.disease ,Cystic fibrosis ,Antiporters ,Solute carrier family ,Evolution, Molecular ,Biochemistry ,Multigene Family ,Biophysics ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Ion transporter ,Function (biology) - Abstract
Transepithelial Cl−and HCO3−transport is critically important for the function of all epithelia and, when altered or ablated, leads to a number of diseases, including cystic fibrosis, congenital chloride diarrhea, deafness, and hypotension ( 78 , 111 , 119 , 126 ). HCO3−is the biological buffer that maintains acid-base balance, thereby preventing metabolic and respiratory acidosis ( 48 ). HCO3−also buffers the pH of the mucosal layers that line all epithelia, protecting them from injury ( 2 ). Being a chaotropic ion, HCO3−is essential for solubilization of ions and macromolecules such as mucins and digestive enzymes in secreted fluids. Most epithelia have a Cl−/HCO3exchange activity in the luminal membrane. The molecular nature of this activity remained a mystery for many years until the discovery of SLC26A3 and the realization that it is a member of a new family of Cl−and HCO3−transporters, the SLC26 family ( 73 , 78 ). This review will highlight structural features, the functional diversity, and several regulatory aspects of the SLC26 transporters.
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- 2008
10. PDZ domain-containing protein as a physiological modulator of TRPV6
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Hyun-Jin Kim, Dongki Yang, and Insuk So
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Amino Acid Motifs ,Molecular Sequence Data ,PDZ domain ,Mutant ,Biophysics ,TRPV Cation Channels ,Plasma protein binding ,Biology ,Biochemistry ,Cell Line ,Protein–protein interaction ,Mice ,Protein structure ,Two-Hybrid System Techniques ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Adaptor Proteins, Signal Transducing ,HEK 293 cells ,Signal transducing adaptor protein ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Electrophysiology ,Gene Expression Regulation ,Mutant Proteins ,Ion Channel Gating ,Protein Binding - Abstract
The epithelial Ca(2+) channel TRPV6 constitutes the apical Ca(2+) entry mechanism in active Ca(2+) transport in kidney and intestine, but little is known about regulation mechanism of TRPV6. We performed yeast two-hybrid screen with TRPV6 C-terminus since TRPV6 has PDZ (Post-synaptic density-95, Drosophila discs-large protein, Zonula occludens protein 1) binding motif at its C terminal end. As a result, we found that 4 PDZ domain-containing protein, PDZK2, interacts with TRPV6 through its fourth PDZ domain. Glutathione S-transferase pull-down assay shows that TRPV6 and PDZK2 directly interact and that TRPV6 C-terminal PDZ binding motif is primarily responsible for this interaction. Mutant Delta4 lacking last 4 amino acid EYQI did not interact with PDZK2. Heterologous overexpression of both TRPV6 and PDZK2 did not show any effect on the activation of TRPV6. On the other hand, the peak current amplitude of mutant Delta4 decreased compared with that of WT TRPV6. When introduced into HEK293 cells expressing TRPV6, PDZ binding motif peptide (EYQI) markedly reduced the peak current amplitude in divalent free (DVF) solution. Knocking down the endogenous PDZK2 of HEK293 cells by RNAi significantly decreased DVF current density. Taken together, we propose that PDZK2 is an essential TRPV6 interacting protein as a physiological modulator of TRPV6.
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- 2007
11. Melastatin-Type Transient Receptor Potential Channel 7 Is Required for Intestinal Pacemaking Activity
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Byung Joo Kim, Insuk So, Chul-Seung Park, Jae Yeoul Jun, Ki Whan Kim, In Youb Chang, Peter R. Stanfield, Hyun Ho Lim, and Dongki Yang
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Male ,Patch-Clamp Techniques ,Cations, Divalent ,Gene Expression ,TRPM Cation Channels ,Biology ,Kidney ,TRPV ,Enteric Nervous System ,Cell Line ,Membrane Potentials ,Mice ,Transient receptor potential channel ,symbols.namesake ,Biological Clocks ,TRPM ,TRPM7 ,Gene expression ,Animals ,Humans ,Cells, Cultured ,TRPC ,Mice, Inbred BALB C ,Hepatology ,Gastroenterology ,Molecular biology ,Interstitial cell of Cajal ,Intestines ,symbols ,Defecation rhythm ,Female ,Gastrointestinal Motility - Abstract
Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut.
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- 2005
12. TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach
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Hyun-Jin Kim, Dongki Yang, Insuk So, Byung Joo Kim, Mei Hong Zhu, Kyu Pil Lee, Ki Whan Kim, and Young Mee Lee
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Male ,Carbachol ,Physiology ,Gene Expression ,Stimulation ,CHO Cells ,GTP-Binding Protein alpha Subunits, Gi-Go ,Muscarinic Agonists ,TRPC5 ,Antibodies ,Membrane Potentials ,Diglycerides ,Mice ,Transient receptor potential channel ,Cations ,Cricetinae ,Physiology (medical) ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Cation Transport Proteins ,TRPC Cation Channels ,Acetylcholine receptor ,Mice, Inbred ICR ,Hepatology ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Anti-Inflammatory Agents, Non-Steroidal ,Stomach ,Gastroenterology ,Muscle, Smooth ,Heterotrimeric GTP-Binding Proteins ,Flufenamic Acid ,Electrophysiology ,Biochemistry ,Gastric Mucosa ,Biophysics ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Calcium ,Female ,Calcium Channels ,Acetylcholine ,medicine.drug - Abstract
We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs+> K+> Na+, similar to that in the murine stomach. With 0.2 mM GTPγS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-Gq/11antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La3+and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.
- Published
- 2003
13. Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1
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Dongki Yang, Hyun Jeong Kwak, Hee-Sook Jun, Yongha Hwang, and Hyae Gyeong Cheon
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Male ,0301 basic medicine ,Physiology ,Palmitates ,Protoporphyrins ,lcsh:Medicine ,Apoptosis ,Pharmacology ,Endoplasmic Reticulum ,Pathology and Laboratory Medicine ,Biochemistry ,Mice ,chemistry.chemical_compound ,Endocrinology ,0302 clinical medicine ,Insulin-Secreting Cells ,Insulin Secretion ,Medicine and Health Sciences ,Insulin ,Small interfering RNAs ,Enzyme-Linked Immunoassays ,Extracellular Signal-Regulated MAP Kinases ,lcsh:Science ,Immune Response ,Endoplasmic Reticulum Chaperone BiP ,Cells, Cultured ,Heat-Shock Proteins ,Secretory Pathway ,Multidisciplinary ,Cell Death ,biology ,Transfection ,Endoplasmic Reticulum Stress ,Nucleic acids ,medicine.anatomical_structure ,Lipotoxicity ,Cell Processes ,Flavanones ,Cytokines ,Scutellaria baicalensis ,RNA Interference ,Cellular Structures and Organelles ,medicine.symptom ,Research Article ,medicine.medical_specialty ,Endocrine Disorders ,Cell Survival ,Immunology ,Inflammation ,DNA Fragmentation ,Research and Analysis Methods ,Protective Agents ,03 medical and health sciences ,Signs and Symptoms ,Diagnostic Medicine ,Internal medicine ,Genetics ,Diabetes Mellitus ,medicine ,Animals ,Non-coding RNA ,Immunoassays ,Diabetic Endocrinology ,Flavonoids ,Biology and life sciences ,Endocrine Physiology ,business.industry ,Pancreatic islets ,lcsh:R ,Cell Biology ,biology.organism_classification ,Hormones ,Gene regulation ,Rats ,Baicalein ,Mice, Inbred C57BL ,Heme oxygenase ,030104 developmental biology ,chemistry ,Metabolic Disorders ,Immunologic Techniques ,Unfolded protein response ,RNA ,Insulinoma ,lcsh:Q ,Gene expression ,business ,Heme Oxygenase-1 ,030217 neurology & neurosurgery - Abstract
Objective beta-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve beta-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on beta-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired beta-cell function, and placed focus on the role of heme oxygenase (HO)-1. Methods Rat pancreatic beta-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 mu M) to induce lipotoxicity in the presence or absence of baicalein (50 mu M), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. Results Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced beta-cell dysfunction. Conclusion Baicalein protects pancreatic beta-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic beta-cells appears to be a promising therapeutic strategy for T2D.
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- 2017
14. Molecular mechanism of pancreatic and salivary gland fluid and HCO3 secretion
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Shmuel Muallem, Dongki Yang, Hyun Woo Park, Ehud Ohana, and Min Goo Lee
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medicine.medical_specialty ,Saliva ,Physiology ,Biology ,Sodium Chloride ,digestive system ,Salivary Glands ,Article ,stomatognathic system ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Humans ,Secretion ,Molecular Biology ,Hco3 secretion ,Pancreas ,Salivary gland ,General Medicine ,Water-Electrolyte Balance ,Cell biology ,Bicarbonates ,medicine.anatomical_structure ,Endocrinology ,Adenomere ,Molecular mechanism ,Function (biology) - Abstract
Fluid and HCO3−secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO3−secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren's syndrome, and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO3−secretion, in particular by secretory glands. Fluid and HCO3−secretion by secretory glands is a two-step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl−and secrete HCO3−. The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete a small amount of NaCl-rich fluid, while the duct absorbs the Cl−and secretes HCO3−and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO3−secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that is driven by active Cl−secretion and contains high concentrations of Na+and Cl−. The salivary glands duct absorbs both the Na+and Cl−and secretes K+and HCO3−. In this review, we focus on the molecular mechanism of fluid and HCO3−secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and to point out the differences to meet gland-specific secretions.
- Published
- 2012
15. Genetic and Pharmacological Inhibition of the Ca2+ Influx Channel TRPC3 Protects Secretory Epithelia from Ca2+-Dependent Toxicity
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Dongki Yang, Shmuel Muallem, Min Seuk Kim, Lutz Birnbaumer, Dong Min Shin, Kyu Pil Lee, Yasuo Mori, Shigeki Kiyonaka, and Joel Abramowitz
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medicine.medical_specialty ,Time Factors ,Salivary Gland Diseases ,Biology ,Severity of Illness Index ,Article ,Salivary Glands ,Mice ,TRPC3 ,stomatognathic system ,Internal medicine ,medicine ,Animals ,Calcium Signaling ,Cell damage ,Pancreas ,TRPC ,Ceruletide ,TRPC Cation Channels ,Mice, Knockout ,Hepatology ,Salivary gland ,Gastroenterology ,Epithelial Cells ,medicine.disease ,Calcium Channel Blockers ,Disease Models, Animal ,Endocrinology ,medicine.anatomical_structure ,Pancreatitis ,Toxicity ,Acute Disease ,Pyrazoles - Abstract
Background & Aims Excessive Ca 2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjogren syndrome. Transient receptor potential (canonical) channel (TRPC) 3 is a major Ca 2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacologic inhibition of TRPC3 protects pancreas and salivary glands from Ca 2+ -dependent damage. Methods We developed a Ca 2+ -dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3 −/− mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3). Results Salivary glands and pancreas of Trpc3 −/− mice were protected from Ca 2+ -mediated cell toxicity. Analysis of Ca 2+ signaling in wild-type and Trpc3 −/− acini showed that Pyr3 is a highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca 2+ -mediated toxicity by inhibiting the Trpc3-mediated component of Ca 2+ influx. Conclusions TRPC3-mediated Ca 2+ influx mediates damage to pancreas and salivary glands. Pharmacologic inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjogren syndrome.
- Published
- 2011
16. Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters
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Dongki Yang, Nikolay Shcheynikov, Insuk So, Ehud Ohana, and Shmuel Muallem
- Subjects
Models, Molecular ,Physiology ,Protein Conformation ,Anion Transport Proteins ,Antiporters ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Protein structure ,Animals ,Humans ,Chloride-Bicarbonate Antiporters ,Structural motif ,Ion transporter ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Ion Transport ,biology ,Permease ,Membrane transport protein ,Transport protein ,Transmembrane domain ,Biochemistry ,Sulfate Transporters ,biology.protein ,Biophysics ,Oocytes ,030217 neurology & neurosurgery - Abstract
Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu(-); Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu(-) in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu(-) charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl(-)/1HCO(3)(-) to 1Cl(-)/1HCO(3)(-), while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.
- Published
- 2011
17. IRBIT: it is everywhere
- Author
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Shmuel Muallem, Dongki Yang, and Nikolay Shcheynikov
- Subjects
inorganic chemicals ,Messenger RNA ,Binding protein ,Protein subunit ,Sodium-Bicarbonate Symporters ,Membrane Proteins ,Transporter ,General Medicine ,Dendritic Cells ,Biology ,Biochemistry ,Article ,Cell biology ,Cellular and Molecular Neuroscience ,Bicarbonates ,Membrane protein ,Animals ,Inositol 1,4,5-Trisphosphate Receptors ,Secretion ,Lectins, C-Type ,Receptor ,Function (biology) - Abstract
IRBIT (IP(3)Rs binding protein released with IP(3)) is a protein originally identified by the Mikoshiba group as an inhibitor of IP(3) receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO(3)(-) transporters to coordinate epithelial HCO(3)(-) secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein.
- Published
- 2010
18. IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway
- Author
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Chou Long Huang, Insuk So, Akihiro Mizutani, Dongki Yang, George Seki, Shmuel Muallem, Katsuhiko Mikoshiba, Qin Li, Hideaki Ando, and Philip Thomas
- Subjects
medicine.medical_specialty ,Cystic Fibrosis Transmembrane Conductance Regulator ,Protein Serine-Threonine Kinases ,Minor Histocompatibility Antigens ,Mice ,WNK Lysine-Deficient Protein Kinase 1 ,Internal medicine ,Protein Phosphatase 1 ,medicine ,SLC26A6 ,Gene silencing ,Animals ,Humans ,Parotid Gland ,Secretion ,Lectins, C-Type ,Gene Silencing ,biology ,Kinase ,urogenital system ,Adenosylhomocysteinase ,Sodium-Bicarbonate Symporters ,Intracellular Signaling Peptides and Proteins ,Pancreatic Ducts ,Membrane Proteins ,Protein phosphatase 1 ,General Medicine ,Cystic fibrosis transmembrane conductance regulator ,Solute carrier family ,Endocrinology ,Gene Expression Regulation ,biology.protein ,Phosphorylation ,Female ,Research Article - Abstract
Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.
- Published
- 2010
19. IRBIT coordinates epithelial fluid and HCO3– secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct
- Author
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Shmuel Muallem, Dongki Yang, Nikolay Shcheynikov, Hideaki Ando, Katsuhiko Mikoshiba, Insuk So, Ehud Ohana, Akihiro Mizutani, and Weizhong Zeng
- Subjects
inorganic chemicals ,Patch-Clamp Techniques ,Cystic Fibrosis Transmembrane Conductance Regulator ,Video microscopy ,Biology ,Antiporters ,Epithelium ,Cell Line ,Mice ,Animals ,Humans ,Secretion ,RNA, Small Interfering ,Receptor ,Epithelial polarity ,Mice, Knockout ,Activator (genetics) ,Binding protein ,Adenosylhomocysteinase ,Sodium-Bicarbonate Symporters ,HEK 293 cells ,Pancreatic Ducts ,Extracellular Fluid ,General Medicine ,respiratory system ,Cell biology ,Bicarbonates ,Biochemistry ,Sulfate Transporters ,Female ,Cotransporter ,Research Article - Abstract
Fluid and HCO3– secretion are vital functions of secretory epithelia. In most epithelia, this entails HCO3– entry at the basolateral membrane, mediated by the Na+-HCO3– cotransporter, pNBC1, and exit at the luminal membrane, mediated by a CFTR-SLC26 transporters complex. Here we report that the protein IRBIT (inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3), a previously identified activator of pNBC1, activates both the basolateral pNBC1 and the luminal CFTR to coordinate fluid and HCO3– secretion by the pancreatic duct. We used video microscopy and ion selective microelectrodes to measure fluid secretion and Cl– and HCO3– concentrations in cultured murine sealed intralobular pancreatic ducts. Short interference RNA–mediated knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, luminal Cl– absorption and HCO3– secretion, and the associated fluid secretion. Single-channel measurements suggested that IRBIT regulated CFTR by reducing channel mean close time. Furthermore, expression of IRBIT constructs in HEK cells revealed that activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, suggesting that IRBIT activates these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO3– secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis.
- Published
- 2008
20. The Slc26a4 transporter functions as an electroneutral Cl-/I-/HCO3- exchanger: role of Slc26a4 and Slc26a6 in I- and HCO3- secretion and in regulation of CFTR in the parotid duct
- Author
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Lawrence P. Karniski, Shmuel Muallem, Susan M. Wall, Weizong Zeng, Insuk So, Youxue Wang, Dongki Yang, and Nikolay Shcheynikov
- Subjects
Physiology ,Xenopus ,Cystic Fibrosis Transmembrane Conductance Regulator ,Biology ,Antiporters ,Xenopus laevis ,In vivo ,SLC26A6 ,otorhinolaryngologic diseases ,Gene silencing ,Animals ,Parotid Gland ,Secretion ,Chloride-Bicarbonate Antiporters ,Transcellular ,Cells, Cultured ,Membrane Transport Proteins ,Transporter ,Hydrogen-Ion Concentration ,biology.organism_classification ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Bicarbonates ,Biochemistry ,Gene Expression Regulation ,Sulfate Transporters ,biology.protein ,Oocytes ,Cellular ,Chlorine ,Iodine ,Signal Transduction - Abstract
Transcellular Cl(-) and HCO(3)(-) transport is a vital function of secretory epithelia and exit across the luminal membrane is mediated by members of the SLC26 transporters in conjunction with cystic fibrosis transmembrane conductance regulator (CFTR) channel. Typically, secretory epithelia express several SLC26 transporters in the same tissue; however, how their specific function is determined in vivo is not known. In the present work we used the parotid gland duct which expressed Slc26a4 and Slc26a6 and the model systems of Slc26a4(-/-) and Slc26a6(-/-) mice to study the role and regulation of these SLC26 transporters. We examined the transport modes of SLC26A4 expressed in Xenopus oocytes and report that SLC26A4 functions as a coupled, electroneutral I(-)/Cl(-), I(-)/HCO(3)(-) and Cl(-)/HCO(3)(-) exchanger with 1: 1 stoichiometry, with I(-) as the preferred anion. In the duct, Slc26a4 is expressed in the luminal membrane and mainly mediates I(-) secretion with minimal role in luminal HCO(3)(-) transport. By contrast, Slc26a6 mediates luminal Cl(-)/HCO(3)(-) exchange activity with minimal role in I(-) secretion. Furthermore, silencing of CFTR altered Cl(-)/HCO(3)(-) exchange by Slc26a6, but had no effect on I(-) secretion by Slc26a4. Accordingly, deletion of Slc26a6, but not deletion of Slc26a4, results in dysregulation of CFTR. These findings provide the first evidence for a selective role of the SLC26 transporters expressed in the same tissue in epithelial anion transport and suggest that transport specificity is achieved by both the properties of the transporters and the composition of the complexes they form.
- Published
- 2008
21. Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells
- Author
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Min Tae Kim, Dong Soo Yeon, Dongki Yang, Byung Joo Kim, Insuk So, Jae Hwa Lee, Seong Chun Kwon, and Ki Whan Kim
- Subjects
Myosin light-chain kinase ,Patch-Clamp Techniques ,Calmodulin ,Physiology ,macromolecular substances ,Cholinergic Agonists ,Kidney ,Cell Line ,Transient receptor potential channel ,Mice ,Animals ,Humans ,Patch clamp ,Enzyme Inhibitors ,RNA, Small Interfering ,Myosin-Light-Chain Kinase ,TRPC ,TRPC Cation Channels ,biology ,HEK 293 cells ,RNA ,Cell Biology ,Cell biology ,Cell culture ,Guanosine 5'-O-(3-Thiotriphosphate) ,biology.protein ,Carbachol - Abstract
The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca2+-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 μM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 μg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPγS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPγS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.
- Published
- 2005
22. Desensitization of canonical transient receptor potential channel 5 by protein kinase C
- Author
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Hyun-Jin Kim, Ki Whan Kim, Insuk So, Nan Ge Jin, MeeRee Chae, Minji Kim, Mei Hong Zhu, Young Mee Lee, and Dongki Yang
- Subjects
Patch-Clamp Techniques ,Physiology ,Cholinergic Agonists ,TRPC5 ,Kidney ,Cell Line ,Membrane Potentials ,Transient receptor potential channel ,Mice ,Adenosine Triphosphate ,Homologous desensitization ,Animals ,Humans ,Patch clamp ,Enzyme Inhibitors ,Phosphorylation ,Cation Transport Proteins ,Egtazic Acid ,TRPC ,Protein kinase C ,Protein Kinase C ,Chelating Agents ,TRPC Cation Channels ,Chemistry ,Cell Biology ,Protein Structure, Tertiary ,Biochemistry ,Mutagenesis ,Biophysics ,Calcium ,Carbachol ,Calcium Channels ,Ion Channel Gating - Abstract
The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca2+-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 μM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 μM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19–36), inhibited this desensitization of TRPC5 activated by 100 μM CCh. When TRPC5 current was activated by intracellular GTPγS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC.
- Published
- 2005
23. Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig
- Author
-
Ju Hong Jeon, Ki Ho Park, Kyung Wan Min, Hyun-Jin Kim, Ki Whan Kim, Dongki Yang, In Gyu Kim, Insuk So, Sung Joon Kim, Kyungho Choi, and Tong Mook Kang
- Subjects
endocrine system ,medicine.medical_specialty ,Carbachol ,Clinical Biochemistry ,Cell ,Guinea Pigs ,Muscarinic Antagonists ,Biology ,Cholinergic Agonists ,Biochemistry ,Ion Channels ,Guinea pig ,Piperidines ,Internal medicine ,Cations ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Drug Interactions ,Receptor ,Molecular Biology ,Base Sequence ,Dose-Response Relationship, Drug ,Stomach ,Muscle, Smooth ,Receptors, Muscarinic ,medicine.anatomical_structure ,Endocrinology ,Gastric Mucosa ,Molecular Medicine ,medicine.symptom ,Acetylcholine ,Muscle contraction ,medicine.drug ,Muscle Contraction - Abstract
Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (I(CCh)) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited I(CCh) dose-dependently with IC(50) of 1.1 0.1 nM (n = 6). GTPgS-induced current, however, was not inhibited by 10 nM 4-DAMP. I(CCh) was not recorded in pertussis-toxin (PTX)-pretreated smooth muscle cells of gastric antrum. I(CCh) values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 3 pA (n = 6) in the control and PTX-treated cells, respectively (P 0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that G(o) protein is important for activation of ACh-activated NSC channels, our results suggest that I(CCh) might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.
- Published
- 2003
24. ATP-sensitive K(+) channels composed of Kir6.1 and SUR2B subunits in guinea pig gastric myocytes
- Author
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Dongki Yang, Tong Mook Kang, Young Chul Kim, Jae Hoon Sim, Insuk So, Sung-Jin Park, and Ki Whan Kim
- Subjects
Male ,medicine.medical_specialty ,Patch-Clamp Techniques ,Potassium Channels ,Physiology ,Protein subunit ,Receptors, Drug ,Vasodilator Agents ,Guinea Pigs ,Gene Expression ,Biology ,Sulfonylurea Receptors ,Guinea pig ,chemistry.chemical_compound ,Adenosine Triphosphate ,Physiology (medical) ,Internal medicine ,Glyburide ,medicine ,Myocyte ,Animals ,Patch clamp ,Potassium Channels, Inwardly Rectifying ,Hepatology ,Pinacidil ,Stomach ,Gastroenterology ,Muscle, Smooth ,Potassium channel ,Cell biology ,Adenosine Diphosphate ,Adenosine diphosphate ,Endocrinology ,chemistry ,Potassium ,Sulfonylurea receptor ,ATP-Binding Cassette Transporters ,Calcium ,Female ,Anti-Arrhythmia Agents - Abstract
This study was designed to identify the single-channel properties and molecular entity of ATP-sensitive K+ (KATP) channels in guinea pig gastric myocytes with patch-clamp recording and RT-PCR. Pinacidil and diazoxide activated KATP currents in a glibenclamide-sensitive manner. The open probability of channels was enhanced by the application of 10 μM pinacidil from 0.085 ± 0.04 to 0.20 ± 0.05 ( n = 7) and was completely blocked by 10 μM glibenclamide. Single-channel conductance was 37.3 ± 2.5 pS ( n = 4) between −80 and −20 mV in symmetrical K+ gradient conditions. In inside-out mode, KATP channels showed no spontaneous openings and were activated by the application of nucleotide diphosphates to the cytoplasmic side. These single-channel properties are similar to those of the nucleotide diphosphate-dependent K+ channels in vascular smooth muscle, which are composed of Kir6.1 and sulfonylurea receptor (SUR)2B. RT-PCR demonstrated the presence of Kir6.1, Kir6.2, and SUR2B in guinea pig stomach smooth muscle cells. These results suggest that KATP channels in smooth muscle cells of the guinea pig stomach are composed of Kir6.1 and SUR2B.
- Published
- 2001
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