Carbon monoxide (CO) is low molecular weight oxide gas that is endogenously produced under physiological conditions and interacts with another gas, nitric oxide (NO), to act as a gastrointestinal messenger. The aim of this study was to determine the effects of exogenous CO on L-type calcium channel currents of human jejunal circular smooth muscle cells. Cells were voltage clamped with 10 mM barium (Ba2+) as the charge carrier, and CO was directly applied into the bath to avoid perfusion induced effects on the recorded currents. 0.2% CO was increased barium current (I (Ba) ) by 15+/-2% (mean+/-S.E., p< 0.01, n=11) in the cells. To determine if the effects of CO on barium current were mediated through the cGMP pathway, cells were pretreated with 1-H-[1, 2, 4]oxadiazolo[4, 3, -a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor, and exogenous CO (0.2%) had no effect on barium currents in the presence of ODQ (2+/-1% increase, n=6, p> 0.05). CO mediates inhibitory neurotransmission through the nitric oxide pathway. Therefore, to determine if the effects of CO on L-calcium channels were also mediated through NO, cells were incubated with N (G) -nitro-L-arginine (L-NNA, 1 mM), a nitric oxide synthase inhibitor. After L-NNA pretreatment, 0.2 % CO did not increase barium current (4+/-2% increase, n=6, p> 0.05). NO donor, SNAP (20 microM) increased barium current by 13+/-2% (n=6, p< 0.05) in human jejunal smooth muscle cells. These data suggest that CO activates L-type calcium channels through NO/cGMP dependant mechanism.
Barium ; Baths ; Calcium Channels, L-Type* ; Carbon Monoxide* ; Carbon* ; Guanylate Cyclase ; Humans* ; Molecular Weight ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nitric Oxide ; Nitric Oxide Synthase ; Perfusion ; Synaptic Transmission ; Tissue Donors

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.

Exogenous carbon monoxide (0.2%) increases L-type calcium (Ca2+) current in human jejunal circular smooth muscle cells. The stimulatory effect of carbon monoxide (CO) on L-type Ca2+ current is inhibited by pre-application of L-NNA, a classical competitive inhibitor of nitric oxide synthase (NOS) with no significant isoform selectivity (Lim, 2003). In the present study, we investigated which isoform of NOS affected CO induced stimulation of L-type Ca2+ current in human jejunal circular smooth muscle cells. Cells were voltage clamped by whole-cell mode patch clamp technique, and membrane currents were recorded with 10 mM barium as the charge carrier. Before the addition of CO, cells were pretreated with each inhibitor of three NOS isoforms for 15 minutes. CO-stimulating effect on L-type Ca2+ current was partially blocked by N- (3- (Amino-methyl) benzyl) acetamidine-2HCl (1400W, an iNOS inhibitor). On the other hand, 3-bromo-7-nitroindazole (BNI, a nNOS inhibitor) or N5- (1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO, an eNOS inhibitor) completely blocked the CO effect. These data suggest that low dose of exogenous CO may stimulate all NOS isoforms to increase L-type Ca2+ channel through nitric oxide (NO) pathway in human jejunal circular smooth muscle cells.
Barium ; Calcium Channels, L-Type ; Calcium* ; Carbon Monoxide ; Carbon* ; Hand ; Humans* ; Membranes ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nitric Oxide Synthase* ; Nitric Oxide* ; Protein Isoforms

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of Ca2+ -activated K+ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward K+ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of K+ current was increased by NS1619, a specific large-conductance Ca2+ -activated K+ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance Ca2+ -activated K+ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various Ca2+ -activated K+ channel modulators were added into culture media for 1~3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance Ca2+ -activated K+ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.
Apamin ; Cell Proliferation ; Clotrimazole ; Culture Media ; Fibroblasts* ; Hand ; Humans* ; Tea ; Wound Healing

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

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*

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