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.

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.

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.

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.

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.

Interactions of Propofol With Human Voltage-gated Kv1.5 Channel Determined by Docking Simulation and Mutagenesis Analyses.

Propofol blocks the voltage-gated human Kv1.5 (hKv1.5) channel by preferentially affecting in its open state. A previous mutational study suggested that several amino acids within the pore region of the hKv1.5 channel are involved in mediating the blocking action of propofol. The present investigation was undertaken to elucidate the predicted binding modes of propofol within the pore cavity of the open-state hKv1.5 channel, using computational docking and mutagenesis approaches. The docking simulation using a homology model of the hKv1.5 channel, constructed based on the crystal structure of the Kv1.2 channel, predicted that propofol was positioned at the base of the pore cavity of hKv1.5 channel, adjacent to 4 amino acids Thr479, Thr480, Val505, and Ile508, and formed arene-H interactions with Val505. The patch-clamp experiments on wild-type and mutant hKv1.5 channels constructed by site-directed mutagenesis revealed that the blocking potency of propofol was significantly reduced in T480A, V505A, and I508A but not in T479A mutants compared with wild-type hKv1.5 channel. These computational docking and experimental mutational analyses suggest that propofol is positioned at the base of the pore cavity and forms functional contact with Thr480, Val505, and Ile508 to directly block the hKv1.5 channel.

Survey of recognition and treatment of at-risk mental state by Japanese psychiatrists.

AIM: The importance of early intervention in psychiatry is widely recognized among psychiatrists. However, it is unknown whether precise knowledge of at-risk mental state has been disseminated. With this survey, we aimed to reveal how Japanese psychiatrists diagnose patients with at-risk mental state and prescribe treatment strategies for them. METHODS: Using fictional case vignettes, we conducted a questionnaire survey of psychiatrists (n = 1399) who worked in Tokyo. We mailed study documents to all eligible participants in November 2015 with a requested return date in December. RESULTS: Two hundred and sixty (19.3%) psychiatrists responded to the survey. Their correct diagnosis rates for the patients in the at-risk mental state vignettes were low (14.6% for the vignette describing at-risk mental state with attenuated positive symptom syndrome; 13.1% for the vignette describing at-risk mental state with brief intermittent psychotic syndrome). Many psychiatrists selected pharmacotherapy and antipsychotics to treat patients in the at-risk mental state vignettes. The psychiatrists who correctly diagnosed patients in the at-risk mental state vignettes had significantly fewer years of clinical psychiatric experience than did those who diagnosed them as having a non-at-risk mental state (12.5 years vs 22.7 years for the vignette describing at-risk mental state with attenuated positive symptom syndrome, P < 0.01; 14.3 years vs 22.2 years for the vignette describing at-risk mental state with brief intermittent psychotic syndrome, P < 0.01). CONCLUSION: This study suggests that precise knowledge of at-risk mental state has not been disseminated among Japanese psychiatrists.

[Successful Case of Coil Embolization for Repeated Pulmonary Arterial Bleeding in Postoperative Empyema with Bronchopleural Fistula].

A 78-year-old woman underwent right S6 segmentectomy and upper lobe partial resection for adenocarcinoma. About 11 months after the operation, she was diagnosed as having empyema with bronchopleural fistula and open thoracotomy was performed. From the following day, active hemorrhage from the pulmonary artery into the thoracic cavity(500~800 ml) repeated. Tamponade, surgical treatment such as putting hemostasis sheet, or covering with a pedicled latissimus dorsi muscle flap could not prevent rebleeding. Therefore selective pulmonary artery coil embolization was performed, after that the rebleeding did not occur.

Direct negative chronotropic action of desflurane on sinoatrial node pacemaker activity in the guinea pig heart.

BACKGROUND: Desflurane inhalation is associated with sympathetic activation and concomitant increase in heart rate in humans and experimental animals. There is, however, little information concerning the direct effects of desflurane on electrical activity of sinoatrial node pacemaker cells that determines the intrinsic heart rate. METHODS: Whole-cell patch-clamp experiments were conducted on guinea pig sinoatrial node pacemaker cells to record spontaneous action potentials and ionic currents contributing to sinoatrial node automaticity, namely, hyperpolarization-activated cation current (If), T-type and L-type Ca currents (ICa,T and ICa,L, respectively), Na/Ca exchange current (INCX), and rapidly and slowly activating delayed rectifier K currents (IKr and IKs, respectively). Electrocardiograms were recorded from ex vivo Langendorff-perfused hearts and in vivo hearts. RESULTS: Desflurane at 6 and 12% decreased spontaneous firing rate of sinoatrial node action potentials by 15.9% (n = 11) and 27.6% (n = 10), respectively, which was associated with 20.4% and 42.5% reductions in diastolic depolarization rate, respectively. Desflurane inhibited If, ICa,T, ICa,L, INCX, and IKs but had little effect on IKr. The negative chronotropic action of desflurane was reasonably well reproduced in sinoatrial node computer model. Desflurane reduced the heart rate in Langendorff-perfused hearts. High concentration (12%) of desflurane inhalation was associated with transient tachycardia, which was totally abolished by pretreatment with the ß-adrenergic blocker propranolol. CONCLUSIONS: Desflurane has a direct negative chronotropic action on sinoatrial node pacemaking activity, which is mediated by its inhibitory action on multiple ionic currents. This direct inhibitory action of desflurane on sinoatrial node automaticity seems to be counteracted by sympathetic activation associated with desflurane inhalation in vivo.

Sevoflurane protects ventricular myocytes against oxidative stress-induced cellular Ca2+ overload and hypercontracture.

BACKGROUND: Oxidative stress is implicated in pathogenesis of cardiac reperfusion injury, characterized by cellular Ca overload and hypercontracture. Volatile anesthetics protect the heart against reperfusion injury primarily by attenuating Ca overload. This study investigated electrophysiological mechanisms underlying cardioprotective effects of sevoflurane against oxidative stress-induced cellular injury. METHODS: The cytosolic Ca levels and cell morphology were assessed in mouse ventricular myocytes, using confocal fluo-3 fluorescence imaging, whereas membrane potentials and L-type Ca current (ICa,L) were recorded using whole-cell patch-clamp techniques. Phosphorylation of Ca/calmodulin-dependent protein kinase II was examined by Western blotting. RESULTS: Exposure to H2O2 (100 µM) for 15 min evoked cytosolic Ca elevation and hypercontracture in 56.8% of ventricular myocytes in 11 experiments, which was partly but significantly reduced by nifedipine, tetracaine, or SEA0400. Sevoflurane prevented H2O2-induced cellular Ca overload in a concentration-dependent way (IC50 = 1.35%). Isoflurane (2%) and desflurane (10%) also protected ventricular myocytes by a degree similar to sevoflurane (3%). Sevoflurane suppressed H2O2-induced electrophysiological disturbances, including early afterdepolarizations, voltage fluctuations in resting potential, and abnormal automaticities. H2O2 significantly enhanced ICa,L by activating Ca/calmodulin-dependent protein kinase II, and subsequent addition of sevoflurane, isoflurane, or desflurane similarly reduced ICa,L to below baseline levels. Phosphorylated Ca/calmodulin-dependent protein kinase II increased after 10-min incubation with H2O2, which was significantly prevented by concomitant administration of sevoflurane. CONCLUSIONS: Sevoflurane protected ventricular myocytes against H2O2-induced Ca overload and hypercontracture, presumably by affecting multiple Ca transport pathways, including ICa,L, Na/Ca exchanger and ryanodine receptor. These actions appear to mediate cardioprotection against reperfusion injury associated with oxidative stress.

Remifentanil has a minimal direct effect on sinoatrial node pacemaker activity in the Guinea pig heart.

BACKGROUND: Whereas remifentanil administration is associated with severe bradycardia, it has yet to be fully investigated whether the negative chronotropic action of remifentanil is mediated by its direct action on sinoatrial (SA) node pacemaker activity in the heart versus indirect results of enhanced vagal activity. METHODS: We examined the effects of remifentanil and fentanyl on the spontaneous action potentials of guinea pig SA node cells at concentrations of 5, 10, 100, and 1000 nM using the amphotericin B-perforated whole-cell patch-clamp technique. Isolated guinea pig hearts were perfused in a Langendorff mode with 5, 10, 100, and 1000 nM remifentanil. RESULTS: The spontaneous firing rate and diastolic depolarization rate (DDR) of the SA node action potentials were 189.1 ± 14.8 /min and 74.1 ± 2.9 mV/s (n = 8), respectively, under control conditions, and were not significantly affected by exposure to 5 nM (P = 1.0 for both spontaneous firing rate and DDR; n = 6), 10 nM (P = 0.62 for spontaneous firing rate, P = 0.99 for DDR; n = 6), or 100 nM (P = 0.23 for spontaneous firing rate, P = 0.38 for DDR; n = 6) remifentanil. However, 1000 nM remifentanil modestly but significantly decreased the spontaneous firing rate (P = 0.0087) and DDR (P = 0.0072, n = 6). Remifentanil did not affect the heart rate of isolated Langendorff-perfused guinea pig hearts at concentrations of 5 nM (P = 0.98), 10 nM (P = 0.35), or 100 nM (P = 0.24) but significantly reduced the heart rate at 1000 nM (P < 0.0001). Fentanyl did not affect the spontaneous firing rate and DDR at concentrations of 5 nM (P = 1.0 for both spontaneous firing rate and DDR) and 10 nM (P = 0.62 for spontaneous firing rate, P = 0.79 for DDR), but it significantly reduced both at 100 nM (P = 0.00038 for spontaneous firing rate, P = 0.0080 for DDR) and 1000 nM (P < 0.0001 for both spontaneous firing rate and DDR). CONCLUSIONS: Clinically relevant concentrations (nanomolar order concentrations) of remifentanil do not produce significant direct effects on intrinsic cardiac automaticity; thus, suggesting that remifentanil-induced bradycardia in the clinical setting is independent of its direct cardiac effects.

Maternal and paternal family history of type 2 diabetes differently influence lipid parameters in young nondiabetic Japanese women.

OBJECTIVES: We assessed the association of family history of type 2 diabetes (T2D) with parameters used for health checkups in young Japanese women. METHODS: The subjects were 497 nondiabetic women aged 19-39 years. Among them, the mothers of 34 subjects and fathers of 50 had T2D (MD group and PD group, respectively). The subjects were assessed for levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG). RESULTS: TC and LDL-C level showed a tendency to increase in the MD group compared with subjects without family history of T2D. LDL-C/HDL-C ratio ≥2.14 was found in 32.4 and 18.0 % of subjects in the MD and PD groups, respectively. When adjusted for differences in age, body mass index, smoking status, and drinking habits, the MD group was found to have a higher risk of abnormal TC and LDL-C levels than the PD group. LDL-C/HDL-C ratio was independently associated with maternal family history but not with paternal family history (odds ratio 3.44 [99 % confidence interval 1.11-10.6] and 1.21 [0.38-3.89], respectively). There was no association between TG/HDL-C ratio and family history type of T2D. CONCLUSIONS: Maternal family history of T2D had a more pronounced effect on the lipid parameters generally evaluated during health checkups than did paternal family history of T2D. Therefore, we recommend systematic screening for early detection and appropriate healthcare guidance for Japanese women, particularly those with maternal family history of T2D.

Abnormality of risk factors for atherosclerotic disease among young Japanese aged 19-39 years old: an evaluation of health checkup data.

OBJECTIVES: Japanese public health policies on the prevention of atherosclerotic diseases have focused on controlling obesity. The aim of this study was to assess the prevalence of abnormality in risk factors for atherosclerotic disease among young Japanese classified according to body mass index (BMI). METHODS: Data were obtained from 359 men (mean age 32 ± 5 years) and 1,108 women (mean age 33 ± 4 years) between 2005 and 2010. Abnormal levels of aspartate aminotransferase, alanine aminotransferase, and/or gamma-glutamyltranspeptidase were considered to indicate liver dysfunction, and abnormal levels of low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and/or triglycerides were considered to indicate dyslipidemia. The cutoff points for high blood pressure (HBP) and hyperglycemia were set as a reference in the high-normal range. RESULTS: No significant difference was observed in both sexes in all items among each year. In men, HBP (12.9 %), liver dysfunction (33.9 %), dyslipidemia (29.9 %), and hyperglycemia (2.7 %) were observed in the normal BMI group (18.5 < BMI ≤ 25 kg/m(2)). In women, these abnormalities were observed in both the normal BMI group and lean group. CONCLUSIONS: Asymptomatic abnormalities, particularly liver dysfunction and dyslipidemia, were observed in a substantial percentage of subjects without obesity. Therefore, we recommend that the comprehensive public health policy should be directed at all individuals, including the non-obese group, for the early prevention/detection against atherosclerotic diseases.

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.

Remifentanil use for cesarean section in a patient with intracranial re-ruptured arteriovenous malformation.

We describe the successful administration of remifentanil as part of the anesthetic technique used for cesarean section performed under general anesthesia in a 24-year-old woman with intracranial re-hemorrhage caused by re-ruptured arteriovenous malformation. A low dose of remifentanil was useful to obtund the hypertensive response during induction and maintenance of anesthesia without neonatal respiratory depression.

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.

Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking the activation of transient receptor potential canonical channels.

BACKGROUND: Volatile anesthetics produce cardioprotective action by attenuating cellular Ca2+ overload. The Ca2+ paradox is an important model for studying the mechanisms associated with Ca2+ overload-mediated myocardial injury, and was recently found to be mediated by Ca2+ entry through the transient receptor potential canonical channels upon Ca2+ repletion. This study investigated the effect of sevoflurane on cellular mechanisms underlying the Ca2+ paradox. METHODS: The Ca2+ paradox was examined in fluo-3 or mag-fluo-4-loaded mouse ventricular myocytes using confocal laser scanning microscope, upon Ca2+ repletion after 15 min of Ca2+ depletion in the absence and presence of sevoflurane. RESULTS: The Ca2+ paradox was evoked in approximately 65% of myocytes upon Ca2+ repletion, as determined by an abrupt elevation of cytosolic Ca2+ accompanied by hypercontracture. The Ca2+ paradox was significantly suppressed by sevoflurane administered for 3 min before and during Ca2+ repletion (Post) or during Ca2+ depletion and repletion (Postlong), and Postlong was more beneficial than Post application. The sarcoplasmic reticulum Ca2+ levels gradually decreased during Ca2+ depletion, and the Ca2+ paradox was readily evoked in myocytes with reduced sarcoplasmic reticulum Ca2+ levels. Postlong but not Post application of sevoflurane prevented decrease in sarcoplasmic reticulum Ca2+ levels by blocking Ca2+ leak through ryanodine receptors. Whole cell patch-clamp recordings revealed that sevoflurane rapidly blocked thapsigargin-induced transient receptor potential canonical currents. CONCLUSIONS: Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking transient receptor potential canonical channels and by preventing the decrease in sarcoplasmic reticulum Ca2+ levels, which is associated with less activation of transient receptor potential canonical channels.