To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K+ currents (IK) of human cardiac fibroblasts (HCFs), we used the wholecell mode patch-clamp technique. Application of CO delivered by carbon monoxidereleasing molecule-3 (CORM3) increased the amplitude of outward K+ currents, and diphenyl phosphine oxide-1 (a specific IK blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester).Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on IK . In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3’s effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced IKactivation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced IK activation. These results suggest that CO enhances IK in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca 2+ -activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents IBK. The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-N G -monomethyl arginine citrate and L-N G -nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with Nethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DLdithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca 2+ -activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents IBK. The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-N G -monomethyl arginine citrate and L-N G -nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with Nethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DLdithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

Although atopic dermatitis (AD) is known to be a representative skin disorder, it also affects the systemic immune response. In a recent study, myoblasts were shown to be involved in the immune regulation, but the roles of muscle cells in AD are poorly understood. We aimed to identify the relationship between mitochondria and atopy by genome-wide analysis of skeletal muscles in mice. We induced AD-like symptoms using house dust mite (HDM) extract in NC/Nga mice. The transcriptional profiles of the untreated group and HDM-induced AD-like group were analyzed and compared using microarray, differentially expressed gene and functional pathway analyses, and protein interaction network construction. Our microarray analysis demonstrated that immune response-, calcium handling-, and mitochondrial metabolism-related genes were differentially expressed. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology pathway analyses, immune response pathways involved in cytokine interaction, nuclear factor-kappa B, and T-cell receptor signaling, calcium handling pathways, and mitochondria metabolism pathways involved in the citrate cycle were significantly upregulated. In protein interaction network analysis, chemokine family-, muscle contraction process-, and immune response-related genes were identified as hub genes with many interactions. In addition, mitochondrial pathways involved in calcium signaling, cardiac muscle contraction, tricarboxylic acid cycle, oxidation-reduction process, and calcium-mediated signaling were significantly stimulated in KEGG and Gene Ontology analyses. Our results provide a comprehensive understanding of the genome-wide transcriptional changes of HDM-induced AD-like symptoms and the indicated genes that could be used as AD clinical biomarkers.
Animals ; Biomarkers ; Calcium ; Calcium Signaling ; Citric Acid ; Citric Acid Cycle ; Cytokines ; Dermatitis, Atopic ; Gene Ontology ; Genome ; Metabolism ; Mice ; Microarray Analysis ; Mitochondria ; Muscle Cells ; Muscle Contraction ; Muscle, Skeletal ; Myoblasts ; Myocardium ; Oxidation-Reduction ; Protein Interaction Maps ; Pyroglyphidae ; Receptors, Antigen, T-Cell ; Skin

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.
Animals ; Biological Processes ; Chemotaxis ; Classification ; Cytokines ; Dermatitis, Contact* ; Gene Expression ; Gene Ontology ; Genome ; Hypersensitivity ; Immune System ; Interleukin-6 ; Mice* ; Models, Animal ; Neutrophils ; Pruritus* ; RNA* ; Sensation ; Sequence Analysis, RNA* ; Signal Transduction ; Skin* ; Transcriptome ; Transient Receptor Potential Channels ; Up-Regulation ; Wound Healing

Despite increased evidence of bio-activity following far-infrared (FIR) radiation, susceptibility of cell signaling to FIR radiation-induced homeostasis is poorly understood. To observe the effects of FIR radiation, FIR-radiated materials-coated fabric was put on experimental rats or applied to L6 cells, and microarray analysis, quantitative real-time polymerase chain reaction, and wound healing assays were performed. Microarray analysis revealed that messenger RNA expressions of rat muscle were stimulated by FIR radiation in a dose-dependent manner in amount of 10% and 30% materials-coated. In 30% group, 1,473 differentially expressed genes were identified (fold change [FC] > 1.5), and 218 genes were significantly regulated (FC > 1.5 and p < 0.05). Microarray analysis showed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and cell migration-related pathways were significantly stimulated in rat muscle. ECM and platelet-derived growth factor (PDGF)-mediated cell migration-related genes were increased. And, results showed that the relative gene expression of actin beta was increased. FIR radiation also stimulated actin subunit and actin-related genes. We observed that wound healing was certainly promoted by FIR radiation over 48 h in L6 cells. Therefore, we suggest that FIR radiation can penetrate the body and stimulate PDGF-mediated cell migration through ECM-integrin signaling in rats.
Actins ; Animals ; Cell Movement* ; Extracellular Matrix ; Focal Adhesions ; Gene Expression ; Homeostasis ; Infrared Rays ; Integrins ; Microarray Analysis ; Muscle, Skeletal* ; Platelet-Derived Growth Factor* ; Rats ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Wound Healing

Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (K(Ca)) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated K(Ca) currents were blocked by iberiotoxin (IbTX, a large conductance K(Ca) blocker). The H2O2-stimulating effect on large-conductance K(Ca) (BK(Ca)) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BK(Ca) currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BK(Ca) currents. Using RT-PCR and western blot analysis, three subtypes of K(Ca) channels were detected in HCFs: BK(Ca) channels, small-conductance K(Ca) (SK(Ca)) channels, and intermediate-conductance K(Ca) (IK(Ca)) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BK(Ca) currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BK(Ca) channels.
Apoptosis ; Blotting, Western ; Cyclic AMP-Dependent Protein Kinases ; Cyclic GMP-Dependent Protein Kinases ; Fibroblasts* ; Guanosine ; Guanylate Cyclase ; Humans* ; Hydrogen Peroxide* ; Hydrogen* ; Oxidative Stress ; Phosphotransferases ; Potassium Channels, Calcium-Activated ; Protein Kinases*

This study surveys the improvement characteristics in old-aged muscular mitochondria by bio-active materials coated fabric (BMCF). To observe the effects, the fabric (10 and 30%) was worn to old-aged rat then the oxygen consumption efficiency and copy numbers of mitochondria, and mRNA expression of apoptosis- and mitophagy-related genes were verified. By wearing the BMCF, the oxidative respiration significantly increased when using the 30% materials coated fabric. The mitochondrial DNA copy number significantly decreased and subsequently recovered in a dose-dependent manner. The respiratory control ratio to mitochondrial DNA copy number showed a dose-dependent increment. As times passed, Bax, caspase 9, PGC-1alpha and beta-actin increased, and Bcl-2 decreased in a dose-dependent manner. However, the BMCF can be seen to have had no effect on Fas receptor. PINK1 expression did not change considerably and was inclined to decrease in control group, but the expression was down-regulated then subsequently increased with the use of the BMCF in a dose-dependent manner. Caspase 3 increased and subsequently decreased in a dose-dependent manner. These results suggest that the BMCF invigorates mitophagy and improves mitochondrial oxidative respiration in skeletal muscle, and in early stage of apoptosis induced by the BMCF is not related to extrinsic death-receptor mediated but mitochondria-mediated signaling pathway.
Actins ; Animals ; Antigens, CD95 ; Apoptosis ; Caspase 3 ; Caspase 9 ; DNA, Mitochondrial ; Mitochondria* ; Mitochondrial Degradation ; Muscle, Skeletal ; Oxygen Consumption ; Rats* ; Respiration ; RNA, Messenger

Taxifolin glycoside is a new drug candidate for the treatment of atopic dermatitis (AD). Many drugs cause side effects such as long QT syndrome by blocking the human ether-a-go-go related gene (hERG) K+ channels. To determine whether taxifolin glycoside would block hERG K+ channels, we recorded hERG K+ currents using a whole-cell patch clamp technique. We found that taxifolin glycoside directly blocked hERG K+ current in a concentration-dependent manner (EC50=9.6+/-0.7 microM). The activation curve of hERG K+ channels was negatively shifted by taxifolin glycoside. In addition, taxifolin glycoside accelerated the activation time constant and reduced the onset of the inactivation time constant. These results suggest that taxifolin glycoside blocks hERG K+ channels that function by facilitating activation and inactivation process.
Dermatitis, Atopic ; Humans ; Long QT Syndrome ; Quercetin

Cardiac fibroblasts constitute one of the largest cell populations in the heart, and contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, their cardiac functions, especially electrophysiological properties, have often been disregarded in studies. Ca2+-activated K+(KCa) channels can control Ca2+influx as well as a number of Ca2+-dependent physiological processes. We, therefore, attempted to identify and characterize KCa channels in rat Cardiac fibroblasts. First, we showed that the cells cultured from the rat ventricle were cardiac fibroblasts by immunostaining for discoidin domain receptor 2 (DDR-2), a specific fibroblast marker. Secondly, we detected the expression of various KCa channels by reverse transcription polymerase chain reaction (RT-PCR), and found all three family members of KCa channels, including large conductance KCa (BK-alpha 1- and -beta 1~4 subunits), intermediate conductance KCa (IK), and small conductance KCa (SK1~4 subunits) channels. Thirdly, we recorded BK, IK, and SK channels by whole cell mode patch clamp technique using their specific blockers. Finally, we performed cell proliferation assay to evaluate the effects of the channels on cell proliferation, and found that the inhibition of IK channel increased the cell proliferation. These results showed the existence of BK, IK, and SK channels in rat ventricular fibroblasts and involvement of IK channel in cell proliferation.
Animals ; Cell Proliferation ; Fibroblasts ; Heart ; Humans ; Myocardium ; Physiological Processes ; Polymerase Chain Reaction ; Rats ; Receptor Protein-Tyrosine Kinases ; Receptors, Mitogen ; Reverse Transcription