Our previous report demonstrated that chick myoblasts are equipped with Ca2+-permeable stretch- activated channels and Ca2+-activated potassium channels (KCa), and that hyperpolarization-induced by KCa channels provides driving force for Ca2+ influx through the stretch-activated channels into the cells. Here, we showed that acetylcholine (ACh) also hyperpolarized the membrane of cultured chick myoblasts, suggesting that nicotinic acetylcholine receptor (nAChR) may be another pathway for Ca2+ influx. Under cell-attatched patch configuration, ACh increased the open probability of KCa channels from 0.007 to 0.055 only when extracellular Ca2+ was present. Nicotine, a nAChR agonist, increased the open probability of KCa channels from 0.008 to 0.023, whereas muscarine failed to do so. Since the activity of KCa channel is sensitive to intracellular Ca2+ level, nAChR seems to be capable of inducing Ca2+ influx. Using the Ca2+ imaging analysis, we were able to provide direct evidence that ACh induced Ca2+ influx from extracellular solution, which was dramatically increased by valinomycin-mediated hyperpolarization. In addition, ACh hyperpolarized the membrane potential from -12.5+/-3 to -31.2+/-5 mV by generating the outward current through KCa channels. These results suggest that activation of nAChR increases Ca2+ influx, which activates KCa channels, thereby hyperpolarizing the membrane potential in chick myoblasts.
Acetylcholine* ; Membrane Potentials ; Membranes ; Muscarine ; Myoblasts* ; Nicotine ; Potassium Channels ; Receptors, Nicotinic

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

Food-dependent exercise-induced anaphylaxis (FDEIA) is a potentially life-threatening clinical reaction in which anaphylaxis develops when physical activity occurs within a few hours after ingesting a specific food. An 18-year-old girl experienced generalized urticaria, periorbital swelling, and dyspnea repeatedly by exercise after intake of a red ginseng health supplement. A confirmed diagnosis was established in this case by using an exercise challenge test after ingesting mixed-plant extract containing red ginseng health supplement. To the best of our knowledge, this is the first case of FDEIA caused by components in a red ginseng health supplement.
Adolescent ; Anaphylaxis* ; Diagnosis ; Dyspnea ; Female ; Humans ; Motor Activity ; Panax ; Plants* ; Urticaria

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

Ca2+ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca2+ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in Ca2+ mobilization of muscle, and that Ca2+ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in Ca2+ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a Ca2+ imaging system, Ca2+ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular Ca2+ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular Ca2+ increase was completely suppressed by treatment of nifedifine, a L-type Ca2+ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular Ca2+ increase in myoblasts and then may play an important role in myoblast fusion.
6-Cyano-7-nitroquinoxaline-2,3-dione ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Kainic Acid* ; Muscle Fibers, Skeletal ; Myoblasts ; Receptors, AMPA ; Receptors, Ionotropic Glutamate ; Receptors, Kainic Acid* ; Receptors, N-Methyl-D-Aspartate

PURPOSE: There is increasing epidemiological evidence of an association between childhood obesity and atopic dermatitis, but little is known about the underlying mechanism(s). In the present study, we used a rat model of atopic dermatitis to assess whether juvenile obesity, induced by reduction of litter size, aggravated the signs of atopic dermatitis and, if so, whether this aggravation was associated with changes in plasma concentration of adipokines, such as leptin and adiponectin. METHODS: Dermatitis was induced by neonatal capsaicin treatment. Body weight, dermatitis score, serum IgE, skin nerve growth factor (NGF), serum leptin and adiponectin, and cytokine mRNA expression in the skin lesion were compared between small (SL, 5 pups) and large litters (LL, 15 pups). RESULTS: The body weight of juvenile rats up to 6 weeks of age was significantly heavier in the SL group, compared with those in the LL group. The SL group showed more robust development of dermatitis, and higher levels of serum IgE and skin NGF than the LL group. Additionally, the SL group demonstrated higher levels of leptin and pro-inflammatory cytokine mRNA but lower levels of adiponectin than the LL group. CONCLUSIONS: These results suggest a causal link between a decrease in immunological tolerance, induced by juvenile obesity, and aggravation of atopic dermatitis.
Adipokines ; Adiponectin ; Animals ; Body Weight ; Capsaicin ; Dermatitis ; Dermatitis, Atopic* ; Immunoglobulin E ; Leptin ; Litter Size ; Models, Animal* ; Nerve Growth Factor ; Obesity* ; Pediatric Obesity ; Plasma ; Rats ; RNA, Messenger ; Skin

PURPOSE: This study was aimed to investigate the relationship between the allergen components and moderate-to-severe allergic reactions in patients with buckwheat allergy. METHODS: Fifteen patients with a history of buckwheat ingestion and a buckwheat specific IgE level> or =0.35 kU/L were enrolled. They were divided into 2 groups according to clinical severity scores, with 0-1 being asymptomatic-to-mild and 2-4 being moderate-to-severe symptoms. Immunoblotting was performed to investigate IgE reactivity toward buckwheat allergens and to measure intensity of each component by using a reflective densitometer. RESULTS: The proportions of positive band to the 16 kDa (62.5% vs 0%, P=0.026) and 40-50 kDa (87.5% vs 28.6%, P=0.041) buckwheat allergens in the grade 2-4 group were higher than those in grade 0-1 group. The level of buckwheat specific IgE of grade 2-4 group was higher than that of grade 0-1 group (41.3 kU/L vs 5.5 kU/L, P=0.037). The median optical densities (ODs) of IgE antibody binding to 40-50 kDa protein were higher in the grade 2-4 group, compared with those in the grade 0-1 group (130% OD vs 60.8% OD, P=0.037). CONCLUSIONS: The 40-50 kDa protein is implicated as an important allergen to predict moderate-to-severe clinical symptoms in Korean children with buckwheat allergy.
Allergens ; Child ; Eating ; Fagopyrum* ; Food Hypersensitivity ; Humans ; Hypersensitivity* ; Immunoblotting ; Immunoglobulin E

PURPOSE: The purpose of this study was to evaluate the utility of specific IgE (sIgE) concentrations for the diagnosis of immediate-type egg and cow's milk (CM) allergies in Korean children and to determine the optimal cutoff levels. METHODS: In this prospective study, children > or =12 months of age with suspected egg or CM allergy were enrolled. Food allergy was diagnosed by an open oral food challenge (OFC) or through the presence of a convincing history after ingestion of egg or CM. The cutoff levels of sIgE for egg white (EW) and CM were determined by analyzing the receiver operating characteristic curves. RESULTS: Out of 273 children, 52 (19.0%) were confirmed to have egg allergy. CM allergy was found in 52 (23.1%) of 225 children. The EW-sIgE concentration indicating a positive predictive value (PPV) of >90% was 28.1 kU/L in children <24 months of age and 22.9 kU/L in those > or =24 months of age. For CM-sIgE, the concentration of 31.4 kU/L in children <24 months of age and 10.1 kU/L in those > or =24 months of age indicated a >90% PPV. EW-sIgE levels of 3.45 kU/L presented a negative predictive value (NPV) of 93.6% in children <24 months of age, while 1.80 kU/L in those > or =24 months of age presented a NPV of 99.2%. The CM-sIgE levels of 0.59 kU/L in children <24 months of age and 0.94 kU/L in those > or =24 months of age showed NPVs of 100% and 96.9%. CONCLUSIONS: Our results indicate that different diagnostic decision points (DDPs) of sIgE levels should be used for the diagnosis of egg or CM allergy in Korean children. The data also suggest that DDPs with high PPV and high NPV are useful for determining whether OFC is required in children with suspected egg or CM allergy.
Child* ; Diagnosis ; Eating ; Egg Hypersensitivity ; Egg White ; Food Hypersensitivity ; Humans ; Hypersensitivity ; Immunoglobulin E* ; Milk ; Milk Hypersensitivity* ; Ovum* ; Prospective Studies ; ROC Curve

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*

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