The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-alpha secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-alpha secretion and NF-kappaB activation. CSE induced an increase in intracellular Ca2+ concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.
Acetylcysteine ; Airway Remodeling ; Asthma ; Bronchioles ; Cell Death ; Cell Movement ; Cell Survival ; Contracts ; Emigration and Immigration ; Humans ; Lung Diseases ; Muscle, Smooth ; Myocytes, Smooth Muscle ; NF-kappa B ; Reactive Oxygen Species ; Smoke ; Tobacco Products ; Tumor Necrosis Factor-alpha

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and > or =25 mm). These follicles had granulosa cell layer and theca interna and the hormone 17beta-estradiol (E2)/ progesterone (P4) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using GeneFishing(TM) DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.
Blotting, Western ; Databases, Nucleic Acid ; Discrimination (Psychology) ; Down-Regulation ; Estrogens ; Female ; Follicular Cyst ; Gene Expression ; Granulosa Cells ; Microtubule-Associated Proteins ; Ovarian Diseases ; Ovary ; Pathologic Processes ; Polymerase Chain Reaction ; Progesterone ; Ribosomal Proteins ; RNA ; Theca Cells ; Up-Regulation

BACKGROUND AND OBJECTIVES: Recent studies have shown that many kinds of K+ channels, including the muscarinic K+ channel (KACh), are activated in the ischemic heart. It is known that these channels can be modulated by phosphorylation. However, little is known about the function of the KACh in ischemic hearts. In this study, we examined whether the KACh channel is mediated by protein kinase C (PKC) activation in rat atrial myocytes under ischemic conditions. MATERIALS AND METHODS: Single atrial cells of adult rat heart were prepared by collagenase digestion. Channel activity of KACh was recorded by cell-attached configuration from single atrial cells under ischemic conditions, using a patch clamp technique. To simulate ischemia, adenosine or potassium cyanide (KCN) was applied to atrial myocytes, and Western blot was performed to specify PKC isoforms. RESULTS: Adenosine and KCN markedly increased KACh channel activity. The responses to adenosine and KCN were increased 3-fold at mean open time from that observed with control. Channel activity of KACh was blocked by pretreatment with PKC antagonists such as sphingosine, Go 6976, and rottlerin. PKC alpha and PKC betaI isoform levels were increased in the membrane fraction of ischemic heart, indicating that ischemic stress might trigger translocation of cytosolic PKC to the cell membrane. CONCLUSION: These results show that KACh channels are modulated by PKC activation under ischemic conditions induced by adenosine or KCN. Therefore, the channels can protect the heart from ischemic stress by increasing channel activity.
Adenosine ; Adult ; Animals ; Blotting, Western ; Cell Membrane ; Collagenases ; Cytosol ; Digestion ; Heart ; Humans ; Ischemia ; Membranes ; Muscle Cells* ; Phosphorylation ; Potassium Cyanide ; Protein Isoforms ; Protein Kinase C* ; Protein Kinases* ; Rats* ; Sphingosine

TWIK-related K+ channel-2 (TREK-2) and TWIK-related spinal cord K+ (TRESK) channel are members of two-pore domain K+ channel family. They are well expressed and help to set the resting membrane potential in sensory neurons. Modulation of TREK-2 and TRESK channels are involved in the pathogenesis of pain, and specifi c activators of TREK-2 and TRESK may be benefi cial for the treatment of pain symptoms. However, the effect of commonly used analgesics on TREK-2 and TRESK channels are not known. Here, we investigated the effect of analgesics on TREK-2 and TRESK channels. The effects of analgesics were examined in HEK cells transfected with TREK-2 or TRESK. Amitriptyline, citalopram, escitalopram, and fluoxetine significantly inhibited TREK-2 and TRESK currents in HEK cells (p<0.05, n=10). Acetaminophen, ibuprofen, nabumetone, and bupropion inhibited TRESK, but had no effect on TREK-2. These results show that all analgesics tested in this study inhibit TRESK activity. Further study is needed to identify the mechanisms by which the analgesics modulate TREK-2 and TRESK differently.
Acetaminophen ; Amitriptyline ; Analgesics* ; Antidepressive Agents* ; Bupropion ; Citalopram ; Fluoxetine ; Humans ; Ibuprofen ; Membrane Potentials ; Potassium Channels, Tandem Pore Domain ; Sensory Receptor Cells ; Spinal Cord

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, alpha-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin beta subunit, and potassium channel tetramerisation domain-containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, alpha-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and alpha-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and alpha-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.
Actins ; Collectins ; Desmin ; Endometritis ; Endometrium ; Female ; Heat-Shock Proteins ; Hemoglobins ; Interleukin-2 ; Potassium Channels ; Proteins ; Proteomics ; Receptors, LH ; RNA, Messenger ; Transferrin

Single-channel recordings of TASK-1 and TASK-3, members of two-pore domain K+ channel family, have not yet been reported in dorsal root ganglion (DRG) neurons, even though their mRNA and activity in whole-cell currents have been detected in these neurons. Here, we report single-channel kinetics of the TASK-3-like K+ channel in DRG neurons and up-regulation of TASK-3 mRNA expression in tissues isolated from animals with spinal cord injury (SCI). In DRG neurons, the single-channel conductance of TASK-3-like K+ channel was 33.0+/-0.1 pS at -60 mV, and TASK-3 activity fell by 65+/-5% when the extracellular pH was changed from 7.3 to 6.3, indicating that the DRG K+ channel is similar to cloned TASK-3 channel. TASK-3 mRNA and protein levels in brain, spinal cord, and DRG were significantly higher in injured animals than in sham-operated ones. These results indicate that TASK-3 channels are expressed and functional in DRG neurons and the expression level is up-regulated following SCI, and suggest that TASK-3 channel could act as a potential background K+ channel under SCI-induced acidic condition.
Acidosis ; Animals ; Brain ; Clone Cells ; Diagnosis-Related Groups ; Ganglia, Spinal ; Humans ; Hydrogen-Ion Concentration ; Kinetics ; Neurons ; Rats ; RNA, Messenger ; Spinal Cord ; Spinal Cord Injuries ; Spinal Nerve Roots ; Up-Regulation

Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain K+ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (~9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.
Animals ; Blotting, Western ; Constriction* ; Diagnosis-Related Groups ; Fluorescent Antibody Technique ; Ganglia, Spinal* ; Humans ; Hydrogen-Ion Concentration ; Mice* ; Neuralgia* ; Neurons ; Polymerase Chain Reaction ; Reverse Transcription ; Riluzole ; RNA, Messenger ; Spinal Nerve Roots* ; Synaptic Transmission

It has been demonstrated that an unidentified cytosolic factor (s) reduces K (ACh) channel function. Therefore, this study attempted to elucidate the cytosolic factor. Fresh cytosol isolated from normal heart (FC) depressed the K (ACh) channel activity, but cytosol isolated from the ischemic hearts (IC) did not modulate the channel function. Electrophorectic analysis revealed that a protein of ~80 kDa was markedly reduced or even lost in IC. By using peptide sequencing analysis and Western blot, this 80 kDa protein was identified as transferrin (receptor-mediated Fe3+ transporter, 76 kDa). Direct application of transferrin (100 nM) to the cytoplasmic side of inside-out patches decreased the open probability (Po, 12.7+/-6.4%, n=4) without change in mean open time (tau o, 98.5+/-1.3%, n=4). However, the equimolar apotransferrin, which is free of Fe3+, had no effect on the channel activity (N*Po, 129.1+/-13.5%, n=3). Directly applied Fe3+ (100 nM) showed results similar to those of transferrin (N*Po: 21.1+/-3.9%, n=5). However Fe2+ failed to reduce the channel function (N*Po, 106.3+/-26.8%, n=5). Interestingly, trivalent cation La (3+) inhibited N*Po of the channel (6.1+/-3.0%, n=3). Taken together, these results suggest that Fe3+ bound to transferrin can modulate the KACh channel function by its electrical property as a polyvalent cation.
Animals ; Blotting, Western ; Cytoplasm ; Cytosol ; Heart ; Muscle Cells* ; Rats* ; Transferrin*

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.
Animals ; Biological Markers/metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; HSP20 Heat-Shock Proteins/metabolism ; HSP27 Heat-Shock Proteins/metabolism ; Microfilament Proteins/metabolism ; Muscle Proteins/metabolism ; Proteome/*biosynthesis ; Proteomics ; Rats ; Rats, Sprague-Dawley ; S100 Proteins/metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord Injuries/*metabolism/pathology ; Urinary Bladder/*metabolism ; *Wound Healing

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitation-contraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than 3 micrometer, diclofenac inhibited reversibly the Na+ current and did irreversibly the L-type Ca2+ channels-mediated inward current (IC50=12.89+/-0.43 micrometer) in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type Ca2+ currents but not the Na+ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type Ca2+ channel, leading to the impairment of E-C coupling in cardiac myocytes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; Calcium Channels, L-Type ; Diclofenac ; Heart ; Heart Failure ; Ibuprofen ; Muscle Cells ; Myocytes, Cardiac ; Naproxen ; Nifedipine ; Patch-Clamp Techniques ; Rats