Prediction of anesthetic torsadogenicity using a human ventricular cell model.

This simulation study was designed to predict the torsadogenicity of sevoflurane and propofol in healthy control, as well as type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), using the O'Hara-Rudy dynamic model. LQT1 and LQT2 models were simulated by decreasing the conductances of slowly and rapidly activating delayed rectifier K+ currents (IKs and IKr, respectively) by 50%, respectively. Action potential duration at 50% repolarization level (APD50) and diastolic intracellular Ca2+ concentration were measured in epicardial cell during administration of sevoflurane (1 ~ 5%) and propofol (1 ~ 10 µM). Torsadogenicity can be predicted from the relationship between APD50 and diastolic intracellular Ca2+ concentration, which is classified by the decision boundary. Whereas the relationships in control and LQT1 models were distributed on nontorsadogenic side in the presence of sevoflurane at all tested concentrations, those in LQT2 models were shifted to torsadogenic side by concentrations of ≥ 2%. In all three models, propofol shifted the relationships in a direction away from the decision boundary on nontorsadogenic side. Our findings suggest that sevoflurane, but not propofol, exerts torsadogenicity in patients with reduced IKr, such as LQT2 patients. Caution should be paid to the occurrence of arrhythmia during sevoflurane anesthesia in patients with reduced IKr.

Severe Calcification of Femoral Arteries Causes Tourniquet Failure Accompanied by Massive Bleeding.

Tourniquet failure is attributed to inadequate tourniquet pressure, inadequate exsanguination, failure to compress medullary vessels within the bone, and incompressible calcified arteries. We herein report a case of massive bleeding using a properly functioning tourniquet in a patient who had bilateral calcified femoral arteries. When incompressible calcified arteries are present, the inflated tourniquet cuff fails to adequately compress the underlying artery, yet acts as an efficient venous tourniquet, which leads to an increase in bleeding. It is therefore critical to preoperatively confirm the effectiveness of the tourniquet in arterial occlusion in patients with severe arterial calcification.

Propofol, an Anesthetic Agent, Inhibits HCN Channels through the Allosteric Modulation of the cAMP-Dependent Gating Mechanism.

Propofol is a broadly used intravenous anesthetic agent that can cause cardiovascular effects, including bradycardia and asystole. A possible mechanism for these effects is slowing cardiac pacemaker activity due to inhibition of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. However, it remains unclear how propofol affects the allosteric nature of the voltage- and cAMP-dependent gating mechanism in HCN channels. To address this aim, we investigated the effect of propofol on HCN channels (HCN4 and HCN2) in heterologous expression systems using a whole-cell patch clamp technique. The extracellular application of propofol substantially suppressed the maximum current at clinical concentrations. This was accompanied by a hyperpolarizing shift in the voltage dependence of channel opening. These effects were significantly attenuated by intracellular loading of cAMP, even after considering the current modification by cAMP in opposite directions. The differential degree of propofol effects in the presence and absence of cAMP was rationalized by an allosteric gating model for HCN channels, where we assumed that propofol affects allosteric couplings between the pore, voltage-sensor, and cyclic nucleotide-binding domain (CNBD). The model predicted that propofol enhanced autoinhibition of pore opening by unliganded CNBD, which was relieved by the activation of CNBD by cAMP. Taken together, these findings reveal that propofol acts as an allosteric modulator of cAMP-dependent gating in HCN channels, which may help us to better understand the clinical action of this anesthetic drug.

Characterization and functional role of rapid- and slow-activating delayed rectifier K+ currents in atrioventricular node cells of guinea pigs.

The atrioventricular (AV) node is the only conduction pathway where electrical impulse can pass from atria to ventricles and exhibits spontaneous automaticity. This study examined the function of the rapid- and slow-activating delayed rectifier K+ currents (IKr and IKs) in the regulation of AV node automaticity. Isolated AV node cells from guinea pigs were current- and voltage-clamped to record the action potentials and the IKr and IKs current. The expression of IKr or IKs was confirmed in the AV node cells by immunocytochemistry, and the positive signals of both channels were localized mainly on the cell membrane. The basal spontaneous automaticity was equally reduced by E4031 and HMR-1556, selective blockers of IKr and IKs, respectively. The nonselective ß-adrenoceptor agonist isoproterenol markedly increased the firing rate of action potentials. In the presence of isoproterenol, the firing rate of action potentials was more effectively reduced by the IKs inhibitor HMR-1556 than by the IKr inhibitor E4031. Both E4031 and HMR-1556 prolonged the action potential duration and depolarized the maximum diastolic potential under basal and ß-adrenoceptor-stimulated conditions. IKr was not significantly influenced by ß-adrenoceptor stimulation, but IKs was concentration-dependently enhanced by isoproterenol (EC50: 15 nM), with a significant negative voltage shift in the channel activation. These findings suggest that both the IKr and IKs channels might exert similar effects on regulating the repolarization process of AV node action potentials under basal conditions; however, when the ß-adrenoceptor is activated, IKs modulation may become more important.

Dexmedetomidine Exerts a Negative Chronotropic Action on Sinoatrial Node Cells Through the Activation of Imidazoline Receptors.

ABSTRACT: Dexmedetomidine (DEX), an α2-adrenoreceptor (α2-AR) and imidazoline receptor agonist, is most often used for the sedation of patients in the intensive care unit. Its administration is associated with an increased incidence of bradycardia; however, the precise mechanism of DEX-induced bradycardia has yet to be fully elucidated. This study was undertaken to examine whether DEX modifies pacemaker activity and the underlying ionic channel function through α2-AR and imidazoline receptors. The whole-cell patch-clamp techniques were used to record action potentials and related ionic currents of sinoatrial node cells in guinea pigs. DEX (≥10 nM) reduced sinoatrial node automaticity and the diastolic depolarization rate. DEX reduced the amplitude of hyperpolarization-activated cation current (If or Ih) the pacemaker current, even within the physiological pacemaker potential range. DEX slowed the If current activation kinetics and caused a significant shift in the voltage dependence of channel activation to negative potentials. In addition, efaroxan, an α2-AR and imidazoline I1 receptor antagonist, attenuated the inhibitory effects of DEX on sinoatrial node automaticity and If current activity, whereas yohimbine, an α2-AR-selective antagonist, did not. DEX did not affect the current activities of other channels, including rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs), L-type Ca2+ current (ICa,L), Na+/Ca2+ exchange current (INCX), and muscarinic K+ current (IK,ACh). Our results indicate that DEX, at clinically relevant concentrations, induced a negative chronotropic effect on the sinoatrial node function through the downregulation of If current through an imidazoline I1 receptor other than the α2-AR in the clinical setting.

Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways.

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both ß1- and ß2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by ß2-adrenoceptor stimulation than ß1-adrenoceptor stimulation. Both ß-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the ß-adrenoceptor-potentiated firing rate. The stimulatory effects of ß- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under ß-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Elevation of propofol sensitivity of cardiac IKs channel by KCNE1 polymorphism D85N.

BACKGROUND AND PURPOSE: The slowly activating delayed rectifier K+ channel (IKs ), composed of pore-forming KCNQ1 α-subunits and ancillary KCNE1 ß-subunits, regulates ventricular repolarization in human heart. Propofol, at clinically used concentrations, modestly inhibits the intact (wild-type) IKs channels and is therefore unlikely to appreciably prolong QT interval in ECG during anaesthesia. However, little information is available concerning the inhibitory effect of propofol on IKs channel associated with its gene variants implicated in QT prolongation. The KCNE1 single nucleotide polymorphism leading to D85N is associated with drug-induced QT prolongation and therefore regarded as a clinically important genetic variant. This study examined whether KCNE1-D85N affects the sensitivity of IKs to inhibition by propofol. EXPERIMENTAL APPROACH: Whole-cell patch-clamp and immunostaining experiments were conducted in HEK293 cells and/or mouse cardiomyocyte-derived HL-1 cells, transfected with wild-type KCNQ1, wild-type or variant KCNE1 cDNAs. KEY RESULTS: Propofol inhibited KCNQ1/KCNE1-D85N current more potently than KCNQ1/KCNE1 current in HEK293 cells and HL-1 cells. Immunostaining experiments in HEK293 cells revealed that pretreatment with propofol (10 µM) did not appreciably affect cell membrane expression of KCNQ1 and KCNE1 proteins in KCNQ1/KCNE1 and KCNQ1/KCNE1-D85N channels. CONCLUSION AND IMPLICATIONS: The KCNE1 polymorphism D85N significantly elevates the sensitivity of IKs to inhibition by propofol. This study detects a functionally important role of KCNE1-D85N polymorphism in conferring genetic susceptibility to propofol-induced QT prolongation and further suggests the possibility that the inhibitory action of anaesthetics on ionic currents becomes exaggerated in patients carrying variants in genes encoding ion channels.

Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion.

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

Feasibility and acceptability of cognitive rehabilitation during the acute phase of schizophrenia.

AIM: Although numerous studies have demonstrated promising results for the cognitive rehabilitation in subjects with schizophrenia, the efficacy of cognitive rehabilitation for everyday and social functioning is not yet sufficient. Although consideration of the contents and methods are vital, the timing for implementing cognitive rehabilitation also seems to be crucial. The aim of this study was to examine the feasibility and acceptability of cognitive rehabilitation during the acute phase of schizophrenia. METHODS: Patients were recruited from consecutive acute admissions to the inpatient unit during a 15-month period and were evaluated to determine whether they could be enrolled in an 8-week cognitive rehabilitation program within 14 days of their hospital admission. Cognitive rehabilitation programs with a workbook style were adopted, taking the patients' conditions and burdens into consideration. RESULTS: Eighty-three patients were newly admitted during the entry period, and 49 patients (59.0%) were eligible for inclusion. Of them, 22 patients (44.9%) agreed to participate and started the program. Sixteen patients completed the program and underwent a second assessment. Thus, 32.7% (16/49) of all the eligible patients actually completed the study. The participants were quite satisfied with the program. CONCLUSIONS: This preliminary study yielded encouraging data demonstrating the feasibility and acceptability of cognitive remediation for patients with schizophrenia during the acute phase. The provision of cognitive rehabilitation during the acute phase of the first episode can reasonably be expected to lead to better functional outcomes.

Effects of motivation domains on social functioning in schizophrenia with consideration of the factor structure and confounding influences.

In patients with schizophrenia, motivation has been assumed to act as an intervening factor between cognitive function and social functioning. Motivation is thought to comprise three domains defined by their orientations: intrinsic motivation, extrinsic motivation, and amotivation. Although these orientations can confound each other, the effects of motivation domains on social functioning remain obscure. We investigated these relationships after confirming the factor structure of social functioning using the Social Functioning Scale (SFS). A total of 97 stable outpatients with schizophrenia were recruited. In addition to the SFS, the General Causality Orientations Scale (GCOS) was used to measure the motivation domains. First, we examined the factor structure of the SFS using exploratory and confirmatory factor analyses. Next, we conducted structural equation modeling to examine the effects of motivation domains on social functioning. The SFS showed a two-factor structure: interpersonal and intrapersonal functioning. The structural equation model revealed that (1) amotivation was negatively related to both intrapersonal and interpersonal functioning, (2) intrinsic motivation was positively related only to intrapersonal functioning, and (3) extrinsic motivation was positively related only to interpersonal functioning. Each motivation domain was associated with different factors of social functioning. Future interventions aimed at improving social functioning should consider these motivation domains based on their orientations. Cognitive remediation accompanied by considerations for and approaches to each of the domains may maximize recovery in patients with schizophrenia.

A computational analysis of the effect of sevoflurane in a human ventricular cell model of long QT syndrome: Importance of repolarization reserve in the QT-prolonging effect of sevoflurane.

The slowly and rapidly activating delayed rectifier K+ channels (IKs and IKr, respectively) contribute to the repolarization of ventricular action potential in human heart and thereby determine QT interval on an electrocardiogram. Loss-of-function mutations in genes encoding IKs and IKr cause type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), accompanied by a high risk of malignant ventricular arrhythmias and sudden cardiac death. This study was designed to investigate which cardiac electrophysiological conditions exaggerate QT-prolonging and arrhythmogenic effects of sevoflurane. We used the O'Hara-Rudy dynamic model to reconstruct human ventricular action potential and a pseudo-electrocardiogram, and simulated LQT1 and LQT2 phenotypes by decreasing conductances of IKs and IKr, respectively. Sevoflurane, but not propofol, prolonged ventricular action potential duration and QT interval in wild-type, LQT1 and LQT2 models. The QT-prolonging effect of sevoflurane was more profound in LQT2 than in wild-type and LQT1 models. The potent inhibitory effect of sevoflurane on IKs was primarily responsible for its QT-prolonging effect. In LQT2 model, IKs was considerably enhanced during excessive prolongation of ventricular action potential duration by reduction of IKr and relative contribution of IKs to ventricular repolarization was markedly elevated, which appears to underlie more pronounced QT-prolonging effect of sevoflurane in LQT2 model, compared with wild-type and LQT1 models. This simulation study clearly elucidates the electrophysiological basis underlying the difference in QT-prolonging effect of sevoflurane among wild-type, LQT1 and LQT2 models, and may provide important information for developing anesthetic strategies for patients with long QT syndrome in clinical settings.

Recreational diving-related injury insurance claims among Divers Alert Network Japan members: Retrospective analysis of 321 cases from 2010 to 2014.

INTRODUCTION: Monitoring trends in diving-related injuries enables implementation of effective safety measures. Divers Alert Network Japan (DAN Japan) membership includes insurance covering recreational diving-related injuries and fatalities. Use of claim data provides both a known denominator and demographic data about injured members. METHODS: The study analysed 325 insurance claims reported to DAN Japan from 2010 to 2014. Four fatalities were excluded, leaving 321 claims for analysis. Claimants were divided into three age groups: young adults (< 40 years); middle-aged (40-59 years) and older adults (≥ 60 years). The total injury claims rate (ICR), decompression illness (DCI) rate (DCR) and trauma rate (TCR) were calculated. Differences between the sexes within each age group were analysed. RESULTS: The total number of DAN Japan member-years in the period was 80,617, with a mean age of 45 years. Claims were made by 153 males and 168 females with a mean and median age of 46 years. Trauma was the most frequent reason for a claim (113 cases, 35%), followed by DCI (109 cases, 34%). The ICR (per 104 member-years) was 39.8 (95% confidence interval 35.5 to 44.2) and the TCR was 14.0 (11.4 to 16.6). For every age group, the ICR and TCR were significantly higher for females than males. The DCR was 13.5 (11.0 to 16.1) and did not significantly differ between the sexes. CONCLUSIONS: The incidence of trauma-related diving injuries exceeds that of claims related to DCI. Females appear to have a higher risk of injury than the general diving population.

Open-channel blocking action of volatile anaesthetics desflurane and sevoflurane on human voltage-gated Kv 1.5 channel.

BACKGROUND AND PURPOSE: Volatile anaesthetics have been shown to differentially modulate mammalian Shaker-related voltage-gated potassium (Kv 1.x) channels. This study was designed to investigate molecular and cellular mechanisms underlying the modulatory effects of desflurane or sevoflurane on human Kv 1.5 (hKv 1.5) channels. EXPERIMENTAL APPROACH: Thirteen single-point mutations were constructed within pore domain of hKv 1.5 channel using site-directed mutagenesis. The effects of desflurane or sevoflurane on heterologously expressed wild-type and mutant hKv 1.5 channels were examined by whole-cell patch-clamp technique. A computer simulation was conducted to predict the docking pose of desflurane or sevoflurane within hKv 1.5 channel. KEY RESULTS: Both desflurane and sevoflurane increased hKv 1.5 current at mild depolarizations but decreased it at strong depolarizations, indicating that these anaesthetics produce both stimulatory and inhibitory actions on hKv 1.5 channels. The inhibitory effect of desflurane or sevoflurane on hKv 1.5 channels arose primarily from its open-channel blocking action. The inhibitory action of desflurane or sevoflurane on hKv 1.5 channels was significantly attenuated in T480A, V505A, and I508A mutant channels, compared with wild-type channel. Computational docking simulation predicted that desflurane or sevoflurane resides within the inner cavity of channel pore and has contact with Thr479, Thr480, Val505, and Ile508. CONCLUSION AND IMPLICATIONS: Desflurane and sevoflurane exert an open-channel blocking action on hKv 1.5 channels by functionally interacting with specific amino acids located within the channel pore. This study thus identifies a novel molecular basis mediating inhibitory modulation of hKv 1.5 channels by desflurane and sevoflurane.

Cigarette Smoking Cessation Temporarily Enhances the Release of Phosphorylated-HSP27 from Human Platelets.

Objective Cigarette smoking is a risk factor for arteriopathy, including acute coronary syndrome, stroke and peripheral vascular disease. Thus, cessation is strongly recommended in order to reduce these risks. We recently demonstrated that smoking cessation causes temporary hyper-aggregability of human platelets. We previously showed that heat shock protein 27 (HSP27) is released from human platelets stimulated by collagen, accompanied by its phosphorylation. Accumulating evidence indicates potent roles of extracellular HSP27 as a modulator of inflammation. In the present study, using the stored samples obtained in the previous study, we investigated the effect of cigarette smoking cessation on the release of phosphorylated-HSP27 from collagen-activated human platelets (n=15 patients). Methods We enrolled patients who visited smoking cessation outpatient services between January 2012 and November 2014. Platelet-rich plasma, chronologically obtained before and after the cessation, was stimulated by collagen using a PA-200 aggregometer in the previous study. The levels of phosphorylated-HSP27 released from platelets were determined by an enzyme-linked immunosorbent assay. The phosphorylation of HSP27 in platelets was evaluated by a Western blot analysis. Results Cessation of cigarette smoking significantly upregulated the levels of collagen-stimulated release of phosphorylated-HSP27 at four and eight weeks after quitting smoking compared to before cessation. However, there was no significant difference between the levels before cessation and those at 12 weeks after cessation. The levels of phosphorylated-HSP27 stimulated by collagen in the platelets at four weeks after smoking cessation were remarkably enhanced compared to before cessation. Conclusion Cigarette smoking cessation temporarily enhances the collagen-stimulated release of phosphorylated-HSP27 from human platelets in the short term.

The release of phosphorylated-HSP27 from activated platelets of obstructive sleep apnea syndrome (OSAS) patients.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is a well known risk of arterial thrombosis that results in cardiovascular morbidity. It has been reported that platelet aggregability is enhanced in patients with OSAS. In the present study, we investigated whether phosphorylated-HSP27 is released from the activated platelets of OSAS patients. METHODS: Patients diagnosed with OSAS (n = 21) were recruited, and platelet-rich plasma (PRP) was stimulated by ADP, ristosetin, collagen, and thrombin receptor-activating peptide. Platelet aggregation was measured using an aggregometer with a laser-scattering system. The levels of protein phosphorylation and the released levels of phosphorylated-HSP27 were determined by Western blot analysis and an ELISA, respectively. RESULTS: The phosphorylation of HSP27 in the platelets was induced by the stimulators. The released levels of phosphorylated-HSP27 was correlated with the levels of phosphorylated-HSP27 stimulated by ADP or collagen. The levels of ADP-induced phosphorylated-HSP27 were correlated with those of both phosphorylated-protein kinase B (Akt) and phosphorylatd-p38 mitogen-activated protein kinase; however, the levels of phosphorylated-HSP27 stimulated by collagen were correlated with phosphorylated-Akt levels only. The ED50 value of ADP on the platelet aggregation in OSAS (1.067 ± 0.128 µM) was lower than that in healthy subjects (1.778 ± 0.122 µM) and was inversely correlated with both the value of minimum SpO2 and the released level of phosphorylated-HSP27 stimulated by ADP. CONCLUSION: The results strongly suggest that phosphorylated-HSP27 is released from the activated platelets of OSAS patients.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

Identification of Verapamil Binding Sites Within Human Kv1.5 Channel Using Mutagenesis and Docking Simulation.

BACKGROUND/AIMS: The phenylalkylamine class of L-type Ca2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium. METHODS: Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore. RESULTS: Verapamil preferentially blocked hKv1.5 channel in its open state with IC50 of 2.4±0.6 µM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516. CONCLUSION: Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans.

Limitation by Rho-kinase and Rac of transforming growth factor-ß-induced interleukin-6 release from astrocytes.

Transforming growth factor (TGF)-ß stimulates release of interleukin (IL)-6, which is recognized to function as both a pro- and anti- inflammatory cytokine in the central nervous system, from astrocytes. It is generally recognized that effects of TGF-ß are mediated through Smad-independent as well as Smad-dependent pathways. Small GTPases regulate a variety of cell functions. In the present study, we investigated whether or not Rho-kinase, a downstream effector of Rho, and Rac are implicated in TGF-ß-stimulated IL-6 release from astrocytes (C8D1A cells). Y-27632 or fasudil (Rho-kinase inhibitors) or NSC23766 (an inhibitor of Rac-guanine nucleotide exchange factor interaction) significantly enhanced TGF-ß-stimulated IL-6 release from these cells. TGF-ß-stimulated IL-6 release was markedly upregulated in RhoA- or Rac-knockdown C8D1A cells. We found that SIS3 (a specific inhibitor of TGF-ß-dependent Smad3 phosphorylation) or LY364947 (a TGF-ß type I receptor kinase inhibitor) significantly reduced the IL-6 release. However, TGF-ß-induced-Smad2 and Smad3 phosphorylation was not affected by Y-27632, fasudil or NSC23766. In conclusion, our results strongly suggest that Rho-kinase and Rac limit TGF-ß-induced IL-6 release from astrocytes, and the suppressive effects are exerted independently of the Smad pathway or at a point downstream of Smad2/3 complex.

Transient Receptor Potential Canonical Channel Blockers Improve Ventricular Contractile Functions After Ischemia/Reperfusion in a Langendorff-perfused Mouse Heart Model.

Reperfusion of ischemic myocardium is accompanied by intracellular Ca overload, leading to cardiac dysfunction. However, the mechanisms underlying intracellular Ca overload have yet to be fully elucidated. The mechanism may involve the activation of store-operated Ca entry, which is primarily mediated through the transient receptor potential canonical (TRPC) channels. This study was undertaken to examine the possible involvement of TRPC channels in the development of contractile dysfunction associated with reperfusion of ischemic myocardium using a mouse heart model. The functional expression of TRPC channels was confirmed in mouse ventricular myocytes using immunocytochemistry, Western blotting, and patch-clamp experiments. The left ventricular functions were assessed by measuring left ventricular end-diastolic pressure, left ventricular developed pressure, and its first derivatives in a Langendorff-perfused mouse heart subjected to 30 minutes of normothermic (37°C) global ischemia followed by 60 minutes of reperfusion. Under control conditions, left ventricular functions were deteriorated during reperfusion, which was significantly ameliorated by administration of the TRPC channel blockers 2-aminoethoxydiphenyl borate and La during initial 5 minutes of reperfusion. Our findings suggest that TRPC channels are involved in mediating contractile dysfunction during reperfusion of ischemic myocardium and detect TRPC channels as a potential therapeutic target for preventing myocardial ischemia/reperfusion injury.