Biomedical subjects

Suk-Ho Lee

Publications and source records attributed to Suk-Ho Lee.

16 recordsLinked to original sources

PubMed2007

Hydrogen peroxide selectively increases TREK-2 currents via myosin light chain kinases.

Two-pore domain K+ (K2P) channels play a critical role in cellular responses to various stimuli, such as stretch or changes in pH and are considered to be important in pathological responses such as apoptosis and tumorigenesis. We investigated effects of H2O2 on various K2P channels expressed in CHO cells. Application of H2O2 did not affect TASK-1, TASK-3, TRAAK currents, but specifically increased TREK-2 currents recorded using a nystatin perforated whole cell technique. The H2O2-induced activation of TREK-2 currents was also observed at single channel levels in cell-attached patches, and the effect was reversed by the reducing agent, dithiothreitol. In contrast, TREK-2 currents recorded using ruptured whole cell technique or single channel recording in inside-out patches were not affected by H2O2. Furthermore, direct application of 5,5'-dithiobis-(2-nitrobenzoic acid) inhibited TREK-2, suggesting that the H2O2-induced activation does not result from direct oxidation of TREK-2 proteins. Among the cell signaling agents, myosin light chain kinase (MLCK) inhibitors significantly inhibited the H2O2-induced activation of TREK-2 currents. These results suggest that TREK-2 channels have a potential to play a specific role in cellular responses to reactive oxygen species and that MLCK activation is involved in this process.

Animals↗

PubMed2006

Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.

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PubMed2006

Postnatal developmental changes in Ca2+ homeostasis in supraoptic magnocellular neurons.

Supraoptic magnocellular neurons (SMNs) undergo dramatic changes in morphological and electrical properties during postnatal development. We investigated the developmental change in Ca2+ homeostasis in SMNs. The decay rate of Ca2+ transients markedly increased during the third postnatal week (PW3) to an adult level. This increase in the Ca2+ decay rate was paralleled by hypertrophy of the SMN somata. Activity of Na+/Ca2+ exchanger (Na/CaX) and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) was quantified as a decrement in the Ca2+ decay rate caused by extracellular [Na+] reduction and that by thapsigargin, respectively. SERCA activity was negligible during PW2, and markedly increased during PW3. SERCA activity and soma size remained stable thereafter. Na/CaX activity was a major Ca2+-clearance mechanism (CCM) during PW2, increased further during PW3, but was negligible in mature SMNs (PW10). In parallel with the decrease in Na/CaX activity, endogenous Ca2+ buffering capacity declined, resulting that the apparent Ca2+ decay rate remained relatively constant between PW4 and PW10. Replacement of intracellular K+ with Li+ had no effect on Na/CaX activity, suggesting that NCX rather than NCKX comprises Na/CaX. These findings indicate a developmental shift in the balance of CCMs from Ca2+ extrusion via NCX toward Ca2+ sequestration into endoplasmic reticulum via SERCA.

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PubMed2006

Dimethyl lithospermate B, an extract of Danshen, suppresses arrhythmogenesis associated with the Brugada syndrome.

BACKGROUND: Dimethyl lithospermate B (dmLSB) is an extract of Danshen, a traditional Chinese herbal remedy, which slows inactivation of INa, leading to increased inward current during the early phases of the action potential (AP). We hypothesized that this action would be antiarrhythmic in the setting of Brugada syndrome. METHODS AND RESULTS: The Brugada syndrome phenotype was created in canine arterially perfused right ventricular wedge preparations with the use of either terfenadine or verapamil to inhibit INa and ICa or pinacidil to activate IK-ATP. AP recordings were simultaneously recorded from epicardial and endocardial sites together with an ECG. Terfenadine, verapamil, and pinacidil each induced all-or-none repolarization at some epicardial sites but not others, leading to ST-segment elevation as well as an increase in both epicardial and transmural dispersions of repolarization (EDR and TDR, respectively) from 12.9+/-9.6 to 107.0+/-54.8 ms and from 22.4+/-8.1 to 82.2+/-37.4 ms, respectively (P<0.05; n=9). Under these conditions, phase 2 reentry developed as the epicardial AP dome propagated from sites where it was maintained to sites at which it was lost, generating closely coupled extrasystoles and ventricular tachycardia and fibrillation. Addition of dmLSB (10 micromol/L) to the coronary perfusate restored the epicardial AP dome, reduced EDR and TDR to 12.4+/-18.1 and 24.4+/-26.7 ms, respectively (P<0.05; n=9), and abolished phase 2 reentry-induced extrasystoles and ventricular tachycardia and fibrillation in 9 of 9 preparations. CONCLUSIONS: Our data suggest that dmLSB is effective in eliminating the arrhythmogenic substrate responsible for the Brugada syndrome and that it deserves further study as a pharmacological adjunct to implanted cardioverter/defibrillator usage.

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PubMed2006

Level set-based bimodal segmentation with stationary global minimum.

In this paper, we propose a new level set-based partial differential equation (PDE) for the purpose of bimodal segmentation. The PDE is derived from an energy functional which is a modified version of the fitting term of the Chan-Vese model. The energy functional is designed to obtain a stationary global minimum, i.e., the level set function which evolves by the Euler-Lagrange equation of the energy functional has a unique convergence state. The existence of a global minimum makes the algorithm invariant to the initialization of the level set function, whereas the existence of a convergence state makes it possible to set a termination criterion on the algorithm. Furthermore, since the level set function converges to one of the two fixed values which are determined by the amount of the shifting of the Heaviside functions, an initialization of the level set function close to those values can result in a fast convergence.

Algorithms↗

PubMed2005

Generation of metabolic oscillations by mitoKATP and ATP synthase during simulated ischemia in ventricular myocytes.

Metabolic oscillations and the concomitant periodic activations of sarcolemmal ATP-sensitive K(+) channels (sarcK(ATP)) have recently been proposed as one mechanism underlying ischemia-related arrhythmia. In this study, we investigated the role of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) and ATP synthase in the generation of metabolic oscillations during simulated ischemia from rat ventricular myocytes using patch-clamp technique and fluorescence microscopy. We have found that the combined application of creatine kinase (CK) inhibitor, 2,4-dinitrofluorobenzene, with cyanide, electron-transport-chain inhibitor causes oscillatory activations of sarcK(ATP). The oscillatory activations of sarcK(ATP) were accompanied by large periodic depolarizations in mitochondrial membrane potential (Psi(m)). 5-Hydroxydecanoate, an inhibitor of mitoK(ATP), halted the oscillations in Psi(m) at repolarized state, whereas oligomycin, an inhibitor of ATP synthase, halted them at depolarized state. In both conditions, oscillatory activations of sarcK(ATP) were abolished. Inhibitors of adenine nucleotide translocator and permeability transition pore had no effect on the oscillations in Psi(m) and sarcK(ATP). 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, an inhibitor of mitochondrial inner-membrane anion channel (IMAC), caused a full depolarization in Psi(m) and activation of sarcK(ATP), finally resulting in irreversible hypercontracture. Taken together, oscillations in Psi(m) can be explained by balance between depolarizing power of mitoK(ATP) and repolarizing power of the reverse activity of ATP synthase. ATP consumption by ATP synthase in reverse mode links periodic depolarizations in Psi(m) to oscillatory activation of sarcK(ATP). Considering that such oscillations were not induced by cyanide alone, CK system may act as an important buffer, inhibiting arrhythmia during ischemia.

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PubMed2005

Interplay between Na+/Ca2+ exchangers and mitochondria in Ca2+ clearance at the calyx of Held.

The clearance of Ca2+ from nerve terminals is critical for determining the build-up of residual Ca2+ after repetitive presynaptic activity. We found previously that K+-dependent Na+/Ca2+ exchangers (NCKXs) show polarized distributions in axon terminals of supraoptic magnocellular neurons and play a major role in Ca2+ clearance. The role of NCKXs in presynaptic terminals, however, has not been studied. We investigated the contribution of NCKX in conjunction with other Ca2+ clearance mechanisms at the calyx of Held by analyzing the decay of Ca2+ transients evoked by depolarizing pulses. Inhibition of Na+/Ca2+ exchange by replacing external Na+ with Li+ decreased the Ca2+ decay rate by 68%. Selective inhibition of NCKX by replacing internal K+ with TEA+ (tetraethylammonium) or Li+ decreased the Ca2+ decay rate by 42%, and the additional inhibition of the K+-independent form of Na+/Ca2+ exchanger (NCX) by reducing external [Na+] caused an additional decrease by 26%. Inhibition of plasma membrane Ca2+-ATPase (PMCA) decreased the Ca2+ decay rate by 23%, whereas inhibition of SERCA (smooth endoplasmic reticulum Ca2+-ATPase) had no effect. The contribution of mitochondria was negligible for small Ca2+ transients but became apparent at [Ca2+]i > 2.5 microM, when Na+/Ca2+ exchange became saturated. Mitochondrial contribution was also observed when the duration of Ca2+ transients was prolonged by inhibiting Na+/Ca2+ exchangers or by increasing Ca2+ buffers. These results suggest that, in response to small Ca2+ transients (<2 microM), Ca2+ loads are cleared from the calyx of Held by NCKX (42%), NCX (26%), and PMCA (23%), and that mitochondria participate when the Ca2+ load is larger or prolonged.

Animals↗

PubMed2005

Harmonic decomposition in PDE-based denoising technique for magnetic resonance electrical impedance tomography.

Recent progress in magnetic resonance electrical impedance tomography (MREIT) research via simulation and biological tissue phantom studies have shown that conductivity images with higher spatial resolution and accuracy are achievable. In order to apply MREIT to human subjects, one of the important remaining problems to be solved is to reduce the amount of the injection current such that it meets the electrical safety regulations. However, by limiting the amount of the injection current according to the safety regulations, the measured MR data such as the z-component of magnetic flux density Bz in MREIT tend to have low SNR and get usually degraded in their accuracy due to the nonideal data acquisition system of an MR scanner. Furthermore, numerical differentiations of the measured Bz required by the conductivity image reconstruction algorithms tend to further deteriorate the quality and accuracy of the reconstructed conductivity images. In this paper, we propose a denoising technique that incorporates a harmonic decomposition. The harmonic decomposition is especially suitable for MREIT due to the physical characteristics of Bz. It effectively removes systematic and random noises, while preserving important key features in the MR measurements, so that improved conductivity images can be obtained. The simulation and experimental results demonstrate that the proposed denoising technique is effective for MREIT, producing significantly improved quality of conductivity images. The denoising technique will be a valuable tool in MREIT to reduce the amount of the injection current when it is combined with an improved MREIT pulse sequence.

Algorithms↗

PubMed2005

Noise removal with gauss curvature-driven diffusion.

In this paper, we propose the use of the Gauss curvature in a Gauss curvature-driven diffusion equation for noise removal. The proposed scheme uses the Gauss curvature as the conductance term and controls the amount of diffusion. The main advantage of the scheme is that it preserves important structures, such as straight edges, curvy edges, ramps, corners, small-scaled features, etc.

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PubMed2004

A novel Na+ channel agonist, dimethyl lithospermate B, slows Na+ current inactivation and increases action potential duration in isolated rat ventricular myocytes.

Voltage-gated Na(+) channel blockers have been widely used as local anaesthetics and antiarrhythmic agents. It has recently been proposed that Na(+) channel agonists can be used as inotropic agents. Here, we report the identification of a natural substance that acts as a Na(+) channel agonist. Using the patch-clamp technique in isolated rat ventricular myocytes, we investigated the electrophysiological effects of the substances isolated from the root extract of Salvia miltiorrhiza, which is known as 'Danshen' in Asian traditional medicine. By the intensive activity-guided fractionation, we identified dimethyl lithospermate B (dmLSB) as the most active component, while LSB, which is the major component of the extract, showed negligible electrophysiological effect. Action potential duration (APD(90)) was increased by 20 microM dmLSB from 58.8 +/- 12.1 to 202.3 +/- 9.5 ms. In spite of the prolonged APD, no early after-depolarization (EAD) was observed. dmLSB had no noticeable effect on K(+) or Ca(2+) currents, but selectively affected Na(+) currents (I(Na)). dmLSB slowed the inactivation kinetics of I(Na) by increasing the proportion of slowly inactivating component without inducing any persistent I(Na). The relative amplitude of slow component compared to the peak fast I(Na) was increased dose dependently by dmLSB (EC(50) = 20 microM). Voltage dependence of inactivation was not affected by dmLSB, while voltage dependence of activation shifted by 5 mV to the depolarised direction. Since the APD prolongation by dmLSB did not provoke EAD, which is thought as a possible mechanism for the proarrhythmia seen in other Na(+) channel agonists, dmLSB might be an excellent candidate for a Na(+) channel agonist.

Action Potentials↗

PubMed2004

Li+ enhances GABAergic inputs to granule cells in the rat hippocampal dentate gyrus.

Defects in GABAergic interneurons are thought to be involved in the pathophysiology of bipolar disorder, and Li+ has been used as a primary therapeutic agent in the treatment. We used the patch clamp technique to investigate whether Li+ affects on spontaneous GABAergic synaptic inputs to granule cells (GCs) in hippocampal dentate gyrus. Extracellularly applied Li+ (25 mM) markedly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs), an effect completely blocked by picrotoxin or bicuculline. Li+ increased sIPSCs frequency in the presence of tetrodotoxin (TTX), but to a lesser extent than its absence. Li+ caused no change in the cumulative amplitude distribution of miniature IPSCs, indicating that a presynaptic mechanism is involved. When TTX was added in the presence of Li+, large-amplitude sIPSCs (>30 pA) were abolished specifically with no effect on small-amplitude sIPSCs (<20 pA). Intracellular Li+ (6 mM) applied via the patch pipette depolarized the resting membrane potential in fast-spiking interneurons, resulting in an increase in spontaneous action potential (AP) firing. This change, however, was not observed in GCs. These results suggest that Li(+)-induced spontaneous AP firing in GABAergic interneurons contributes to the increase in GABAergic synaptic inputs to GCs.

Action Potentials↗

PubMed2003

Distribution of K+-dependent Na+/Ca2+ exchangers in the rat supraoptic magnocellular neuron is polarized to axon terminals.

Neurons are polarized into compartments such as the soma, dendrites, and axon terminals, each of which has highly specialized functions. To test whether Ca2+ is differently handled in different compartments of a neuron, we investigated Ca2+ clearance mechanisms in somata of supraoptic magnocellular neurosecretory cells (MNCs) and in their axon terminals located in neurohypophyses. Using patch-clamp and microfluorometry techniques, Ca2+ transients were evoked by depolarizing pulses. Endogenous Ca2+ binding ratios (kappaS) and Ca2+ clearance rates were calculated from the decay phases of Ca2+ transients according to the single compartment model. Mean values of kappaS were 79 +/- 2.6 in somata of MNCs and 187 +/- 19 in axon terminals. Ca2+ clearance rate in axon terminals, which were calculated from time derivative of Ca2+ decay and the kappaS values, were approximately threefold higher than in somata. In response to external Na+ reduction, Ca2+ clearance rates were reduced by 65% in axon terminals, but did not change in somata. Immunohistochemical assays confirmed that K+-dependent Na+/Ca2+ exchanger (NCKX2) was specifically localized to neurohypophysial axon terminals and was not found in somata. In somata, inhibition of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pumps, mitochondrial Ca2+-uniporter, and plasma membrane Ca2+-ATPase (PMCA) pumps decreased Ca2+ clearance rate by 48, 27, and 21%, respectively. These results suggest that neurohypophysial axon terminals have greater Ca2+ clearance power than somata because of the specific localization of NCKX2, and that Ca2+ clearance in somata of MNCs is mediated by SERCA pumps, mitochondrial uniporter, and PMCA pumps.

Animals↗

PubMed2003

Role of K(+) channels in frequency regulation of spontaneous action potentials in rat pituitary GH(3) cells.

The frequency of spontaneous action potentials (SAP) is important in the regulation of hormone secretion. The decrease in K(+) conductance is known as a primary mechanism for increasing SAP frequency. To investigate the nature of K(+) channels that contribute to the frequency regulation of the SAP in rat clonal pituitary GH(3) cells, the effect of various K(+) channel blockers on the SAP and membrane currents were recorded using the patch-clamp technique. A classical inward rectifying K(+) channel blocker, Cs(+) (5 mM), caused an increase in firing frequency and depolarization in after-hyperpolarization (AHP) voltage. An ETHER-A-GO-GO(ERG) type K(+) channel blocker, E-4031 (5 microM), caused no significant effect on the SAP. Tetraethylammonium (TEA, 10 mM) decreased firing frequency and increased the duration of SAP. These effects were not changed by the presence of high concentration of Ca(2+) buffer (10 mM EGTA or BAPTA) in pipette solutions. In voltage-clamp experiments, Cs(+) and E-4031 did not affect outwardly rectifying K(+) currents, but significantly inhibited inwardly rectifying K(+) currents recorded in isotonic K(+) solution. However, the kinetics of Cs(+)-sensitive current and E-4031-sensitive current were distinctive: the time to peak was more immediate and the decay rate was slower in Cs(+)-sensitive current than in E-4031-sensitive current. These results imply that Cs(+) and E-4031 inhibit the distinct components of inwardly rectifying K(+) currents, and that the contribution of the Cs(+)-sensitive current can be immediate on repolarization and can last more effectively over pacemaking potential range than E-4031-sensitive current.

Action Potentials↗

PubMed2002

K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis.

Two different families of Na+/Ca2+ exchangers, K+-independent NCX and K+-dependent NCKX, are known. Exploiting the outward K+ gradient, NCKX is able to extrude Ca2+ more efficiently than NCX, even when the Na+ gradient is reduced. The NCKX, which was originally thought to be limited to the retinal photoreceptor, was shown recently to be widely distributed in the brain. We investigated the contribution of Na+/Ca2+ exchange to Ca2+ clearance mechanisms in neurohypophysial (NHP) axon terminals, using patch-clamp and microfluorometry techniques. In the presence of internal K+, Ca2+ decay was significantly slowed by the removal of external Na+, indicative of the role of Na+/Ca2+ exchange. As internal [K+] was decreased, Ca2+ decay rate and its dependence on Na+ were greatly attenuated. In the absence of internal K+, Ca2+ decay rate was little affected by Na+ removal. Quantitative analysis using Ca2+ decay rate constant indicated that >60% of Ca2+ extrusion is mediated by Na+/Ca2+ exchange when peak [Ca2+] level is higher than 500 nm, and approximately 90% of Na+/Ca2+ exchange activity is K+ dependent. In situ hybridization confirmed the expression of NCKX2 transcripts in the supraoptic nucleus in which soma of NHP axon terminals are located. To our knowledge, this is the first report to show the significant role of K+-dependent Na+/Ca2+ exchange in neuronal cells other than photoreceptors. Considering that axon terminals are subject to an invasion by high-frequency Na+ spikes, which may lower Na+ gradients, the presence of NCKX may have a functional significance in intracellular Ca2+ regulation.

Animals↗

PubMed2002

Monitoring of ANP secretion from single atrial myocytes using densitometry.

Atrial myocytes secrete atrial natriuretic peptide (ANP) in response to mechanical stretch and can serve as a challenging model for studying stretch-secretion coupling. We have developed a technique for monitoring ANP secretion from single atrial myocytes, using neutral red and a CCD video camera. Atrial-specific granules (ASGs) containing ANP were stained with neutral red. The cells were illuminated with monochromatic light (550 mm) and the grey value monitored within the region of interest (ROI) surrounding the region in which ASGs were densely located. Assuming that neutral red is evenly distributed in ASGs, the change in optical density (OD) was considered to represent the total amount of secretion. Under control, non-stimulated conditions, the OD decreased spontaneously (19.7+/-1.4%/10 min, n=14). Direct mechanical stretch (cell length increased by 20%) with two micropipettes or hypotonic swelling (200 mOsm) accelerated the decrease in OD significantly (48.7+/-7.4%/10 min; n=3, 47.2+/-2.4%/10 min; n=7, respectively). In conclusion, this method allows monitoring of ANP secretion with a relatively high time resolution while mechanical stress is applied. Furthermore, patch-clamp or intracellular perfusion techniques can be combined with the present technique for studying cellular mechanisms of stretch-secretion coupling.

Animals↗

PubMed2002

Intracellular Mg(2+) hyperpolarizes rabbit coronary artery smooth muscle cells by differential modulation of Ca(2+)(i)-dependent ion channels.

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (> or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.

Animals↗