Effects of rosiglitazone, an antidiabetic drug, on Kv3.1 channels

Rosiglitazone is a thiazolidinedione-class antidiabetic drug that reduces blood glucose and glycated hemoglobin levels. We here investigated the interaction of rosiglitazone with Kv3.1 expressed in Chinese hamster ovary cells using the wholecell patch-clamp technique. Rosiglitazone rapidly and reversibly inhibited Kv3.1 currents in a concentration-dependent manner (IC 50 = 29.8 µM) and accelerated the decay of Kv3.1 currents without modifying the activation kinetics. The rosiglitazonemediated inhibition of Kv3.1 channels increased steeply in a sigmoidal pattern over the voltage range of –20 to +30 mV, whereas it was voltage-independent in the voltage range above +30 mV, where the channels were fully activated. The deactivation of Kv3.1 current, measured along with tail currents, was also slowed by the drug. In addition, the steady-state inactivation curve of Kv3.1 by rosiglitazone shifts to a negative potential without significant change in the slope value. All the results with the use dependence of the rosiglitazone-mediated blockade suggest that rosiglitazone acts on Kv3.1 channels as an open channel blocker.

The antidiabetic drug rosiglitazone blocks Kv1.5 potassium channels in an open state

An antidiabetic drug, rosiglitazone is a member of the drug class of thiazolidinedione. Although restrictions on use due to the possibility of heart toxicity have been removed, it is still a drug that is concerned about side effects on the heart. We here examined, using Chinese hamster ovary cells, the action of rosiglitazone on Kv1.5 channels, which is a major determinant of the duration of cardiac action potential. Rosiglitazone rapidly and reversibly inhibited Kv1.5 currents in a concentrationdependent manner (IC 50 = 18.9 µM) and accelerated the decay of Kv1.5 currents without modifying the activation kinetics. In addition, the deactivation of Kv1.5 current, assayed with tail current, was slowed by the drug. All of the results as well as the usedependence of the rosiglitazone-mediated blockade indicate that rosiglitazone acts on Kv1.5 channels as an open channel blocker. This study suggests that the cardiac side effects of rosiglitazone might be mediated in part by suppression of Kv1.5 channels, and therefore, raises a concern of using the drug for diabetic therapeutics.

Antidepressant drug paroxetine blocks the open pore of Kv3.1 potassium channel

In patients with epilepsy, depression is a common comorbidity but difficult to be treated because many antidepressants cause pro-convulsive effects. Thus, it is important to identify the risk of seizures associated with antidepressants. To determine whether paroxetine, a very potent selective serotonin reuptake inhibitor (SSRI), interacts with ion channels that modulate neuronal excitability, we examined the effects of paroxetine on Kv3.1 potassium channels, which contribute to highfrequency firing of interneurons, using the whole-cell patch-clamp technique. Kv3.1 channels were cloned from rat neurons and expressed in Chinese hamster ovary cells. Paroxetine reversibly reduced the amplitude of Kv3.1 current, with an IC₅₀ value of 9.43 µM and a Hill coefficient of 1.43, and also accelerated the decay of Kv3.1 current. The paroxetine-induced inhibition of Kv3.1 channels was voltage-dependent even when the channels were fully open. The binding (k₊₁) and unbinding (k₋₁) rate constants for the paroxetine effect were 4.5 µM⁻¹s⁻¹ and 35.8 s⁻¹, respectively, yielding a calculated K(D) value of 7.9 µM. The analyses of Kv3.1 tail current indicated that paroxetine did not affect ion selectivity and slowed its deactivation time course, resulting in a tail crossover phenomenon. Paroxetine inhibited Kv3.1 channels in a usedependent manner. Taken together, these results suggest that paroxetine blocks the open state of Kv3.1 channels. Given the role of Kv3.1 in fast spiking of interneurons, our data imply that the blockade of Kv3.1 by paroxetine might elevate epileptic activity of neural networks by interfering with repetitive firing of inhibitory neurons.

Blockade of Kv1.5 by paroxetine, an antidepressant drug

Paroxetine, a selective serotonin reuptake inhibitor (SSRI), has been reported to have an effect on several ion channels including human ether-a-go-go-related gene in a SSRI-independent manner. These results suggest that paroxetine may cause side effects on cardiac system. In this study, we investigated the effect of paroxetine on Kv1.5, which is one of cardiac ion channels. The action of paroxetine on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Paroxetine reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 4.11 microM and 0.98, respectively. Paroxetine accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to 0 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.32. The binding (k(+1)) and unbinding (k(-1)) rate constants for paroxetine-induced block of Kv1.5 were 4.9 microM(-1)s(-1) and 16.1 s-1, respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 3.3 microM. Paroxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of paroxetine, were superimposed. Inhibition of Kv1.5 by paroxetine was use-dependent. The present results suggest that paroxetine acts on Kv1.5 currents as an open-channel blocker.

Blockade of Kv1.5 channels by the antidepressant drug sertraline

Sertraline, a selective serotonin reuptake inhibitor (SSRI), has been reported to lead to cardiac toxicity even at therapeutic doses including sudden cardiac death and ventricular arrhythmia. And in a SSRI-independent manner, sertraline has been known to inhibit various voltage-dependent channels, which play an important role in regulation of cardiovascular system. In the present study, we investigated the action of sertraline on Kv1.5, which is one of cardiac ion channels. The eff ect of sertraline on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Sertraline reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 0.71 microM and 1.29, respectively. Sertraline accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -20 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +10 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.16. Sertraline slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of sertraline, were superimposed. Inhibition of Kv1.5 by sertraline was use-dependent. The present results suggest that sertraline acts on Kv1.5 currents as an open-channel blocker.

Inhibitory Actions of HERG Currents by the Immunosuppressant Drug Cyclosporin A

The effect of cyclosporin A (CsA), an immunosuppressant, on human ether-a-go-go-related gene (HERG) channel as it is expressed in human embryonic kidney cells was studied using a whole-cell, patch-clamp technique. CsA inhibited the HERG channel in a concentration-dependent manner, with an IC50 value and a Hill coefficient of 3.17 microM and 0.89, respectively. Pretreatment with cypermethrine, a calcineurin inhibitor, had no effect on the CsA-induced inhibition of the HERG channel. The CsA-induced inhibition of HERG channels was voltage-dependent, with a steep increase over the voltage range of the channel opening. However, the inhibition exhibited voltage independence over the voltage range of fully activated channels. CsA blocked the HERG channels predominantly in the open and inactivated states rather than in the closed state. Results of the present study suggest that CsA acts directly on the HERG channel as an open-channel blocker, and it acts independently of its effect on calcineurin activity.

Open Channel Block of Kv3.1 Currents by Genistein, a Tyrosine Kinase Inhibitor

The goal of this study was to analyze the effects of genistein, a widely used tyrosine kinase inhibitor, on cloned Shaw-type K+ currents, Kv3.1 which were stably expressed in Chinese hamster ovary (CHO) cells, using the whole-cell configuration of patch-clamp techniques. In whole-cell recordings, genistein at external concentrations from 10 to 100 micrometer accelerated the rate of inactivation of Kv3.1 currents, thereby concentration-dependently reducing the current at the end of depolarizing pulse with an IC50 value of 15.71+/-0.67 micrometer and a Hill coefficient of 3.28+/-0.35 (n=5). The time constant of activation at a 300 ms depolarizing test pulses from -80 mV to +40 mV was 1.01+/-0.04 ms and 0.90+/-0.05 ms (n=9) under control conditions and in the presence of 20 micrometer genistein, respectively, indicating that the activation kinetics was not significantly modified by genistein. Genistein (20 micrometer) slowed the deactivation of the tail current elicited upon repolarization to -40 mV, thus inducing a crossover phenomenon. These results suggest that drug unbinding is required before Kv3.1 channels can close. Genistein-induced block was voltage-dependent, increasing in the voltage range (-20 mV~0 mV) for channel opening, suggesting an open channel interaction. Genistein (20 micrometer) produced use-dependent block of Kv3.1 at a stimulation frequency of 1 Hz. The voltage dependence of steady-state inactivation of Kv3.1 was not changed by 20 micrometer genistein. Our results indicate that genistein blocks directly Kv3.1 currents in concentration-, voltage-, time-dependent manners and the action of genistein on Kv3.1 is independent of tyrosine kinase inhibition.

Effect of Genistein, a Tyrosine Kinase Inhibitor, on the Cloned Rat Brain Potassium Channel Kv1.5

The effect of genistein, widely used as a specific tyrosine kinase inhibitor, on rat brain Kv1.5 channels which were stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Genistein inhibited Kv1.5 currents at +50 mV in a concentration-dependent manner, with an IC50 of 54.7+/-8.2 micrometer and a Hill coefficient of 1.1+/-0.2. Pretreatment of Kv1.5 with protein tyrosine kinase inhibitors (10 micrometer lavendustin A and 100 micrometer AG1296) and a tyrosine phosphatase inhibitor (500 micrometer sodium orthovanadate) did not block the inhibitory effect of genistein. The inhibition of Kv1.5 by genistein showed voltage-independence over the full activation voltage range positive to 0 mV. The activation (at +50 mV) kinetics was significantly delayed by genistein: time constant for an activation of 1.4+/-0.2 msec under control conditions and 10.0+/-0.5 msec in the presence of 60 micrometer genistein. Genistein also slowed the deactivation of the tail currents, resulting in a crossover phenomenon: a time constant of 11.4+/-1.3 msec and 40.0+/-4.2 msec under control conditions and in the presence of 60 micrometer genistein, respectively. Inhibition was reversed by the application of repetitive depolarizing pulses, especially during the early part of the activating pulse. These results suggest that genistein directly inhibits Kv1.5 channels, independent of phosphotyrosine-signaling pathway.

Direct Block of Cloned K+ Channels, Kv1.5 and Kv1.3, by Cyclosporin A, Independent of Calcineurin Inhibition

The interaction of cyclosporine A (CsA), an immunosuppressant, with rat brain Kv1.5 (Kv1.5) channels, which were stably expressed in Chinese hamster ovary cells, was investigated using the whole-cell patch-clamp technique. CsA reversibly blocked Kv1.5 currents at +50 mV in a reversible concentration- dependent manner with an apparent IC50 of 1.0microM. Other calcineurin inhibitors (cypermethrin, autoinhibitory peptide) had no effect on Kv1.5 and did not prevent the inhibitory effect of CsA. Fast application of CsA led to a rapid and reversible block of Kv1.5, and the onset time constants of the CsA-induced block were decreased in a concentration-dependent manner. The CsA-induced block of Kv1.5 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the block exhibited voltage independence over the voltage range in which channels were fully activated. The rate constants for association and dissociation of CsA were 7.0microM-1s-1 and 8.1 s-1, respectively. CsA slowed the deactivation time course, resulting in a tail crossover phenomenon. Block of Kv1.5 by CsA was use-dependent. CsA also blocked Kv1.3 currents at +50 mV in a reversible concentration-dependent manner with an apparent IC50 of 1.1microM. The same effects of CsA on Kv1.3 were also observed in excised inside-out patches when applied to the internal surface of the membrane. The present results suggest that CsA acts directly on Kv1.5 currents as an open-channel blocker, independently of the effects of CsA on calcineurin activity.

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

Age-related decline in expression of calnexin

Aging is accompanied by the changes in the cells that decrease their capacity to respond to various forms of stress. Cells are known to respond to stresses through expression of stress- response proteins, heat-shock proteins composed of molecular chaperones. Recent studies suggest that chaperone level and stress-induced chaperone expression could decrease with aging. The aim of the present study is to identify chaperones that show a significant change in protein expression with aging. We used an in vitro aging model system of human diploid fibroblasts (HDF). Proteome analysis of HDF showed that endoplasmic reticulum (ER) chaperone, calnexin, significantly decreased with aging. Oxidative stress-induced expression of calnexin also attenuated in old HDF compared to young cells. These findings suggest calnexin decreases with aging and might contribute to a cytoprotection in a variety of human age-related diseases.

Surveillance of Surgical Wound Infections among Patients from the Department of Surgery: Prospective Trial

PURPOSE: Despite advances in infection control practices, Surgical Site Infections (SSIs) remain a substantial cause of morbidity and mortality among hospitalized patients. This study was undertaken to determine prospectively the incidence of postoperative wound infections in surgical patients and to identify the risk factors associated with the development of wound infections. METHODS: Prospective data on 761 surgical operation patients in the department of surgery at Ewha Womans University Mokdong Hospital were collected over a 7 month-period from May 1, to December 31, 2001. The Centers for Disease Control and Prevention (CDC)'s definitions of surgical wound infections were used. RESULTS: A total of 761 patients were observed over 30 days. The overall incidence of wound infection was 2%. SSIs were significantly associated with the degree of wound contamination (P=0.0004). The infection rate increased as the degree of wound contamination increased from clean (1.4%) through clean-contaminated (1.8%) and contaminated (1.8%), to dirty- infected wound (12.7%). The infection rate was related with the American Society of Anesthesiologists (ASA) preoperative assessment score (P=0.00153). There were no SSIs from laparoscopic surgery. The duration of operation was not associated with an increase in wound infections. Enterococcus faecium and Staphylococcus aureus were the most frequently isolated organisms. Three out of the five (60%) cases of E. faecium were vancomycin-resistant enterococci (VRE) and all of three cases of S. aureus were methicillin- resistant Staphylococcus aureus (MRSA). CONCLUSION: This study confirms that the degree of wound contamination is a significant preoperative risk factor for SSI. Many antibiotic-resistant bacteria such as MRSA and VRE were isolated. Accordingly, infection control practitioners need to consider this risk factor in the design of effective infection control strategies. There should be another safe and feasible option available for the treatment of selective patients.

Vibrio vulnificus Cytolysin Forms Anion-selective Pores on the CPAE Cells, a Pulmonary Endothelial Cell Line

Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of 37.5 4.0 pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of Na in bath solution with monovalent ions (K, Cs or TEA ) or with divalent ions (Mg2, Ca2 ) did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to 11.8 and 28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at 40 mV was Cl = NO2 > or = Br = I > SCN > acetate > isethionate > ascorbic acid > EDTA2, in descending order. The cytolysin-induced pore current was blocked by Cl channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells.

Semiquantitative Measurement of Hepatitis B Virus e Antigen by Elecsys 2010 Immunoanalyzer in Lamivudine Treated Patients / 대한진단검사의학회지

BACKGROUND: These days the Hepatitis B virus (HBV) DNA quantitation is the common tool for defining the viral replication state and antiviral therapeutic effect. However, it has a slow turn around time due to a labour intensive complex procedure. The HBeAg assay is a simple, rapid test, but it gives only qualitative information. However, Elecsys 2010 immunoanalyzer (Roche Diag-nostics GmbH, Germany) gives not only qualitative information but also semiquantitative information. So, we want to know about the clinical utility of HBeAg semiquantitation on Elecsys 2010. METHODS: HBeAg and HBV DNA were measured serially for 18 months by Elecsys 2010 and the Hybrid Capture System (HCS; Digene Corporation, Gaithersburg, USA) respectively to evaluate their general correlations (n=287) and serial-changing patterns after lamivudine treatment (n=30, 12-18 months) in 41 patients positive for HBeAg and HBV DNA. RESULTS: Positive correlation was found between the semiquantitative results of HBeAg and the quantitative results of HBV DNA (R=0.56, P<0.05). At two and five months after lamivudine treatment, significant differences (P<0.05) between HBV DNA disappearance groups (group 2, 3) and the HBV DNA remnant group (group 4) were found in the HBeAg level and a decreasing rate. The serial HBeAg and HBV DNA level and the decreasing rate showed a similar pattern in the HBV DNA disappearance groups (group 2, 3). However, a markedly delayed decreasing pattern in the HBeAg level was observed in the HBV DNA remnant group, when compared with HBV DNA. CONCLUSIONS: HBeAg semiquantitation using Elecsys 2010 would provide a cost effective, fast and valuable, and supplementary monitoring tool when the result might be reported in quantity in a dynamic range, because HBeAg semiquantitation could provide an outline of the HBV replication state HBeAg positive patients and a therapeutic predictive value in HBeAg positive, lamivudine treated patients.

Clinical Pathways for Acute Appendicitis: Approach for DRG

BACKGROUND: The current health care system demands provision of quality patient care in a cost-effective manner. A clinical path defines an optimal sequencing and timing of intervention by a health care team. This path facilitates the streamlining of this process. Implementation of clinical paths may decrease hospital cost without increasing complications in acute appendicitis patients. METHODS: A prospective evaluation of a clinical pathway for acute appendicitis (during March 1999) was conducted and the results were compared with those for control patients (during Feb 1999). Pregnant patients or patients with chronic disease were excluded. The patients with acute appendicitis were classified into three groups: A-type for acute focal and suppurative appendicitis, B-type for gangrenous appendicitis, and C-type for perforative appendicitis. RESULTS: The data for 40 patients with a clinical pathway were compared to those for 30 control patients. The mean age was 25.3 11.7 years in the pathway group versus 39.3 15.8 years in the control group. The mean hospital duration were 4.5 days for the pathway with A-type appendicitis versus 5 days for the control patients (p<0.05) and the mean hospital cost was 85.73% of that for the control group (p<0.05). In B- and C-type, the hospital duration and the cost were not different. The satisfaction rates were increased in all the types of pathway patients. The complication rates for in all the pathways were no different from those for the control patients. CONCLUSION: The clinical pathway with A-type appendicitis decreased the duration of hospitalization and the cost without adversely affecting the diagnosis or the therapy. The clinical paths were useful as means to minimize cost while increasing patient satisfaction.