KIOM-79, a mixture of ethanol extracts from four herbs (parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix), has been developed for the potential therapeutic application to diabetic symptoms. Because screening of unexpected cardiac arrhythmia is compulsory for the new drug development, we investigated the effects of KIOM-79 on the action potential (AP) and various ion channel currents in cardiac myocytes. KIOM-79 decreased the upstroke velocity (Vmax) and plateau potential while slightly increased the duration of action potential (APD). Consistent with the decreased Vmax and plateau potential, the peak amplitude of Na+ current (INa) and Ca2+ current (ICa,L) were decreased by KIOM-79. KIOM-79 showed dual effects on hERG K+ current; increase of depolarization phase current (Idepol) and decreased tail current at repolarization phase (Itail). The increase of APD was suspected due to the decreased Itail. In computer simulation, the change of cardiac action potential could be well simulated based on the effects of KIOM-79 on various membrane currents. As a whole, the influence of KIOM-79 on cardiac ion channels are minor at concentrations effective for the diabetic models (0.1-10 microg/mL). The results suggest safety in terms of the risk of cardiac arrhythmia. Also, our study demonstrates the usefulness of the cardiac computer simulation in screening drug-induced long-QT syndrome.
Action Potentials/*drug effects ; Animals ; Cell Line ; Computer Simulation ; Female ; Ginger/chemistry ; Humans ; Ion Channels/*physiology ; Long QT Syndrome/diagnosis ; Male ; Membrane Potentials/drug effects/physiology ; Myocytes, Cardiac/*drug effects/physiology ; Patch-Clamp Techniques ; Plant Extracts/*pharmacology ; Pueraria/chemistry ; Purkinje Fibers/drug effects/physiology ; Rabbits ; Rats ; Rats, Sprague-Dawley

KIOM-79, a mixture of ethanol extracts from four herbs (parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix), has been developed for the potential therapeutic application to diabetic symptoms. Because screening of unexpected cardiac arrhythmia is compulsory for the new drug development, we investigated the effects of KIOM-79 on the action potential (AP) and various ion channel currents in cardiac myocytes. KIOM-79 decreased the upstroke velocity (Vmax) and plateau potential while slightly increased the duration of action potential (APD). Consistent with the decreased Vmax and plateau potential, the peak amplitude of Na+ current (INa) and Ca2+ current (ICa,L) were decreased by KIOM-79. KIOM-79 showed dual effects on hERG K+ current; increase of depolarization phase current (Idepol) and decreased tail current at repolarization phase (Itail). The increase of APD was suspected due to the decreased Itail. In computer simulation, the change of cardiac action potential could be well simulated based on the effects of KIOM-79 on various membrane currents. As a whole, the influence of KIOM-79 on cardiac ion channels are minor at concentrations effective for the diabetic models (0.1-10 microg/mL). The results suggest safety in terms of the risk of cardiac arrhythmia. Also, our study demonstrates the usefulness of the cardiac computer simulation in screening drug-induced long-QT syndrome.
Action Potentials/*drug effects ; Animals ; Cell Line ; Computer Simulation ; Female ; Ginger/chemistry ; Humans ; Ion Channels/*physiology ; Long QT Syndrome/diagnosis ; Male ; Membrane Potentials/drug effects/physiology ; Myocytes, Cardiac/*drug effects/physiology ; Patch-Clamp Techniques ; Plant Extracts/*pharmacology ; Pueraria/chemistry ; Purkinje Fibers/drug effects/physiology ; Rabbits ; Rats ; Rats, Sprague-Dawley

Since first discovered in chick skeletal muscles, stretch-activated channels (SACs) have been proposed as a probable mechano-transducer of the mechanical stimulus at the cellular level. Channel properties have been studied in both the single-channel and the whole-cell level. There is growing evidence to indicate that major stretch-induced changes in electrical activity are mediated by activation of these channels. We aimed to investigate the mechanism of stretch-induced automaticity by exploiting a recent mathematical model of rat atrial myocytes which had been established to reproduce cellular activities such as the action potential, Ca2+ transients, and contractile force. The incorporation of SACs into the mathematical model, based on experimental results, successfully reproduced the repetitive firing of spontaneous action potentials by stretch. The induced automaticity was composed of two phases. The early phase was driven by increased background conductance of voltage-gated Na+ channel, whereas the later phase was driven by the reverse-mode operation of Na+/Ca2+ exchange current secondary to the accumulation of Na+ and Ca2+ through SACs. These results of simulation successfully demonstrate how the SACs can induce automaticity in a single atrial myocyte which may act as a focus to initiate and maintain atrial fibrillation in concert with other arrhythmogenic changes in the heart.
Action Potentials ; Animals ; Atrial Fibrillation ; Fires ; Heart ; Models, Theoretical ; Muscle Cells ; Muscle, Skeletal ; Rats

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca2+](c) of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe< or =5.5) activated outwardly rectifying Cl- current (I(Cl,pH)) with slow kinetics of voltage-dependent activation. I(Cl,pH) was potently inhibited by an anion channel blocker 4,4`-diisothiocyanostilbene-2,2`-disulphonic acid (DIDS, 73.5% inhibition at 1micrometer). I(Cl,pH) became more sensitive to pHe by raising temperature from 24degrees C to 37degrees C. HaCaT cells also expressed Ca2+ -activated Cl- current (I(Cl,Ca)), and the amplitude of I(Cl,Ca) was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca2+] (delta[Ca2+](acid)) of HaCaT cells. The delta[Ca2+](acid) was independent of extracellular Ca2+, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed delta[Ca2+](acid). In summary, we found I(Cl,pH) and delta[Ca2+](acid) in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.
Cell Line ; Estrenes ; Foreskin ; Humans ; Hydrogen-Ion Concentration ; Keratinocytes ; Kinetics ; Membranes ; Pyrrolidinones ; Skin

No abstract available.
B-Lymphocytes*

The intermediate conductance Ca2+-activated K+ channels (SK4, IKCa1) are present in lymphocytes, and their membrane expression is upregulated by various immunological stimuli. In this study, the activity of SK4 was compared between Bal-17 and WEHI-231 cell lines which represent mature and immature stages of murine B lymphocytes, respectively. The whole-cell patch clamp with high-Ca2+ (0.8microM) KCl pipette solution revealed a voltage-independent K+ current that was blocked by clotrimazole (1 mM), an SK4 blocker. The expression of mRNAs for SK4 was confirmed in both Bal-17 and WEHI-231 cells. The density of clotrimazole-sensitive SK4 current was significantly larger in Bal-17 than WEHI-231 cells (-11.4+/-3.1 Vs. -5.7+/-1.15 pA/pF). Also, the chronic stimulation of B cell receptors (BCR) by BCR-ligation (anti-IgM Ab, 3microgram/ml, 8~12 h) significantly upregulated the amplitude of clotrimazole-sensitive current from -11.4+/-3.1 to -53.1+/-8.6 pA/pF in Bal-17 cells. In WEHI-231 cells, the effect of BCR-ligation was significantly small (-5.7+/-1.15 to -9.0+/-1.00 pA/pF). The differential expression and regulation by BCR-ligation might reflect functional changes in the maturation of B lymphocytes.
B-Lymphocytes* ; Cell Line ; Clotrimazole ; Lymphocytes ; Membranes ; Potassium Channels ; Potassium Channels, Calcium-Activated ; RNA, Messenger

TRPM7, a cation channel protein permeable to various metal ions such as Mg2+, is ubiquitously expressed in variety of cells including lymphocytes. The activity of TRPM7 is tightly regulated by intracellular Mg2+, thus named Mg2+-inhibited cation (MIC) current, and its expression is known to be critical for the viability and proliferation of B lymphocytes. In this study, the level of MIC current was compared between immature (WEHI-231) and mature (Bal-17) B lymphocytes. In both cell types, an intracellular dialysis with Mg2+-free solution (140 mM CsCl) induced an outwardly-rectifying MIC current. The peak amplitude of MIC current and the permeability to divalent cation (Mn2+) were several fold higher in Bal-17 than WEHI-231. Also, the level of mRNAs for TRPM7, a molecular correspondence of the MIC channel, was significantly higher in Bal-17 cells. The amplitude of MIC was further increased, and the relation between current and voltage became linear under divalent cation-free conditions, demonstrating typical properties of the TRPM7. The stimulation of B cell receptors (BCR) by ligation with antibodies did not change the amplitude of MIC current. Also, increase of extracellular [Mg2+]c to enhance the Mg2+ influx did not affect the BCR ligation-induced death of WEHI-231 cells. Although the level of TRPM7 was not directly related with the cell death of immature B cells, the remarkable difference of TRPM7 might indicate a fundamental change in the permeability to divalent cations during the development of B cells.
Antibodies ; B-Lymphocytes* ; Cations, Divalent ; Cell Death ; Dialysis ; Ions ; Ligation ; Lymphocytes ; Permeability ; Precursor Cells, B-Lymphoid ; RNA, Messenger

We developed a cardiac cell model to explain the phenomenon of mechano-electric feedback (MEF), based on the experimental data with rat atrial myocytes. It incorporated the activity of ion channels, pumps, exchangers, and changes of intracellular ion concentration. Changes in membrane excitability and Ca2+ transients could then be calculated. In the model, the major ion channels responsible for the stretch-induced changes in electrical activity were the stretch-activated channels (SACs). The relationship between the extent of stretch and activation of SACs was formulated based on the experimental findings. Then, the effects of mechanical stretch on the electrical activity were reproduced. The shape of the action potential (AP) was significantly changed by stretch in the model simulation. The duration was decreased at initial fast phase of repolarization (AP duration at 20% repolarization level from 3.7 to 2.5 ms) and increased at late slow phase of repolarization (AP duration at 90% repolarization level from 62 to 178 ms). The resting potential was depolarized from -75 to -61 mV. This mathematical model of SACs may quantitatively predict changes in cardiomyocytes by mechanical stretch.
Action Potentials ; Animals ; Ion Channels ; Membrane Potentials ; Membranes ; Models, Theoretical ; Muscle Cells* ; Myocytes, Cardiac ; Rats*

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance Ca2+ -activated K+ channel (BKCa) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of BKCa. The BKCa currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single BKCa currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit BKCa currents in the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent K+ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.
4-Aminopyridine* ; Charybdotoxin ; Discrimination (Psychology) ; Ion Channels ; Membrane Potentials ; Muscle, Smooth* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle* ; Patch-Clamp Techniques ; Pulmonary Artery

L-type Ca2+ channels play an important role in regulating cytosolic Ca2+ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type Ca2+ channel currents in rat pituitary GH3 cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the Ba2+ current through the L-type Ca2+ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the Ba2+ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced Ba2+ currents. The above results show that the L-type Ca2+ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in GH3 cells may be attributable, at least in part, to their effect on L-type Ca2+ channels.
Adenylyl Cyclases ; Animals ; Cell Line* ; Colforsin ; Cytosol ; Genistein ; Neuroendocrine Cells ; Nucleotides, Cyclic ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; Phosphotransferases ; Protein Kinase C ; Protein Kinases* ; Protein Tyrosine Phosphatases ; Protein-Tyrosine Kinases ; Rats* ; Vanadates