Inactivation Constant Research Articles

Asenapine, an atypical antipsychotic, blocks voltage-gated potassium channels in rabbit coronary artery smooth muscle cells

We investigated the effect of asenapine, a commonly used atypical antipsychotic, on voltage-dependent K+ (Kv) channels in rabbit coronary artery smooth muscle cells. Asenapine inhibited the Kv current in a concentration-dependent manner, with an half-inhibitory concentration (IC50) value of 8.59 ± 2.25 μM and Hill coefficient of 0.64 ± 0.06. Although asenapine did not affect the steady-state activation curve of Kv channels, it shifted the voltage dependence of the steady-state inactivation curve toward a more negative potential. Asenapine increased the recovery time constant of channel inactivation and produced use (state)-dependent inhibition of Kv channels at a stimulation frequency of 1 or 2 Hz. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 reduced the Kv current; however, additional application of asenapine did not further inhibit the Kv current. Pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and Kv7 inhibitor linopirdine also reduced the Kv current. However, additional application of asenapine further reduced the Kv current, similar to the application of asenapine alone. Asenapine induced membrane depolarization and vasoconstriction. Based on these results, we conclude that asenapine inhibits the Kv current in concentration- and use (state)-dependent manners by shifting the inactivation curve. The major target of asenapine is the Kv1.5 subtype channel.

Effect of rice flour ultrafine particle size on β-glucanase inactivation by microwave treatments and pasting properties in treated flours

The main problem in developing gluten-free products enriched with β-glucans (BG) is the hydrolysis they suffer due to the endogenous β-glucanase content of raw materials. The depolymerization causes a decrease in the molecular weight and viscosity, which is the most important parameter related to the health claims of BG. Hence, it is necessary to inactivate the β-glucanase. Therefore, this study aimed to inactivate the β-glucanase activity present in rice flour (Indica variety) with fine particle size using the microwave (MW) hydrothermal treatment method. The rice flour was conditioned at three different humidities 13, 16, 19 and 25%. Samples of 50 g were subjected to different microwave treatments 1, 2, 4 and 8 min, at 900 W power at 20 seconds intervals of treatment and 1-minute rest. The effect of particle size on inactivating the β-glucanase activity and the effect of MW treatment on the flour pasting property were also studied. The inactivation process followed a first-order kinetic response, and the apparent rate constant of thermal inactivation increased exponentially with the moisture content (M) of the flour, according to the equation -0.02t·exp (0.19·M) (R2 = 0.987). Among the treatments employed 4 min treatment at 25% initial M was found effective for complete β-glucanase inactivation. The effect of flour particle size did not show a significant (p> 0.05) difference on the efficiency of β-glucanase inactivation. Keywords: particle size, β-glucans, rice flour, microwave treatment, β-glucanase activity.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor(STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STBachieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25ºC and 37ºC (0.128 and 0.148h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Blockade of Voltage-Dependent K+ Channels by Benztropine, a Muscarinic Acetylcholine Receptor Inhibitor, in Coronary Arterial Smooth Muscle Cells.

We investigated the effect of the acetylcholine muscarinic receptor inhibitor benztropine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Benztropine inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 6.11 ± 0.80 μM and Hill coefficient of 0.62 ± 0.03. Benztropine shifted the steady-state activation curves toward a more positive potential, and the steady-state inactivation curves toward a more negative potential, suggesting that benztropine inhibited Kv channels by affecting the channel voltage sensor. Train pulse (1 or 2 Hz)-induced Kv currents were effectively reduced by the benztropine treatment. Furthermore, recovery time constants of Kv current inactivation increased significantly in response to benztropine. These results suggest that benztropine inhibited vascular Kv channels in a use (state)-dependent manner. The inhibitory effect of benztropine was canceled by pretreatment with the Kv 1.5 inhibitor, but there was no obvious change after pretreatment with Kv 2.1 or Kv7 inhibitors. In conclusion, benztropine inhibited the Kv current in a concentration- and use (state)-dependent manner. Inhibition of the Kv channels by benztropine primarily involved the Kv1.5 subtype. Restrictions are required when using benztropine to patients with vascular disease.

Kinetics and mechanism of ozonation to treat Microcystis-laden source waters affected by cell-viability

Toxic cyanobacteria are challenging drinking water safety globally, and their cell-viability declines at decay stage of a succussive bloom. Ozone might be a more effective oxidant to treat both high- and low-viability cyanobacteria than other common oxidants (e.g., chlorine, potassium permanganate). However, previous studies only conducted ozonation experiments using high-viability cyanobacteria, and potential influences of cell-viability on ozonation process, remains unknown. In this study, kinetics of ozone decay, cell inactivation, membrane destruction, and cyanotoxin fate of high- and low-viability Microcystis (the most common genus), was investigated, and associated mechanism was discussed. Results showed that low-viability Microcystis exhibited a higher rate constant of membrane destruction (665–744 M−1 s−1) than high-viability Microcystis (364–600 M−1 s−1) by equal concentrations of ozone, ascribed to loosely gelatinous sheath comprised with fewer organic matters as oxidant scavengers. Meanwhile, a higher rate constant of photosynthetic inactivation induced by ozonation, was observed for low-viability Microcystis (312–364 M−1 s−1) than that for high-viability Microcystis (168–294 M−1 s−1). However, elevated aromatic organics competitively inhibited microcystin ozonation for low-viability Microcystis, and hydroxyl radicals for microcystin oxidation could be reduced by elevated organic loads and alkalinity. Moreover, elevated ozone exposure (>51 mg min L−1) did not totally oxidize microcystin with a residual of 30 μg L−1 for low-viability Microcystis. These findings suggested that elevated microcystin risk would be the great barrier to limit ozonation application for low-viability Microcystis, even with benefits of higher cell inactivation compared to high-viability Microcystis.

Inactivation of plant pathogenic bacterium Ralstonia solanacearum in drainage solution from hydroponic system by a rotating advanced oxidation contactor equipped with TiO2/zeolite composite sheets

A rotating advanced oxidation contactor (RAOC) equipped with TiO2/zeolite composite sheets was developed to inactivate the plant pathogenic bacterium Ralstonia solanacearum in drainage solution (DS) from a hydroponic system. The inactivation efficiency of R. solanacearum in DS and pure culture solution (PS) by the RAOC was compared with that achieved by a submerged composite sheet photocatalysis reactor (SSPR). The initial number of living bacteria (N0) was adjusted to around 106–107 CFU mL−1. The inactivation efficiency of R. solanacearum by the SSPR at 4.0 × 107 CFU mL−1 of N0 significantly decreased compared with that at 1.8 × 106 CFU mL−1 of N0 owing to the attenuation of UV intensity by light absorption and scattering by solids derived from R. solanacearum, while the RAOC achieved >2-log inactivation during 24 h of treatment regardless of N0. The inactivation of R. solanacearum by the RAOC decelerated after 6 h, possibly because of competition for reactive oxygen species between R. solanacearum and products accumulated by inactivation of R. solanacearum. The ratio of rate constants for inactivation of R. solanacearum in DS to that in PS by the RAOC was 8 times that for the SSPR. This shows that the RAOC greatly mitigates the light attenuation and inhibitory effects of coexisting substances on inactivation of R. solanacearum in the DS. The RAOC is therefore a promising and upscalable photocatalytic reactor for efficient inactivation of R. solanacearum in DS.

An efficient and scalable data analysis solution for automated electrophysiology platforms.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

The dual effect of shellac on survival of spray-dried Lactobacillus rhamnosus GG microcapsules

Heat shock and hygroscopicity are two main factors that resulted in low viability of probiotics in spray-dried microcapsules during storage. Hydrophobic polyester shellac was combined with whey protein isolate (WPI) to solve this problem. The results suggested that although the survival rate after drying decreased from 20.63% to 0.01% with increased shellac to WPI ratio, the 1:1 shellac-WPI provided the best protection among all samples during storage. The consistence between moisture-adsorption-isotherm and bacterial inactivation constants confirmed the moisture barrier effect of shellac under moderate humidity. Single-droplet drying and differential scanning calorimeter revealed that shellac addition reduced the drying rate and glass transition temperature of microcapsules, which in turn decreased the membrane integrity and growth capability of the probiotics after drying. This study revealed the dual effect of hydrophobic material on instant and long-term survival of spray-dried probiotic microcapsules, which provided new sight to the design of composite wall materials.

Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V

ABSTRACT JZTX-V is a toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. Previous studies had shown that JZTX-V could inhibit the transient outward potassium current of Kv4.2 and Kv4.3 expressed in Xenopus oocytes but had no effects on Kv1.2–1.4. However, the underlying action mechanism of JZTX-V on Kv4.3 remains unclear. In our study, JZTX-V could inhibit not only transient outward potassium currents evoked in small-sized DRG neurons but also Kv4.3-encoded currents expressed in HEK293T cells in the concentration and voltage dependence. The half maximal inhibitory concentration of JZTX-V on Kv4.3 was 9.6 ± 1.2 nM. In addition, the time course for JZTX-V inhibition and release of inhibition after washout were 15.8 ± 1.54 s and 58.8 ± 4.35 s. Electrophysiological assays indicated that 25 nM JZTX-V could shift significantly the voltage dependence of steady-state activation and steady-state inactivation to depolarization. Meanwhile, 25 nM JZTX-V decreased markedly the time constant of activation and inactivation but had no effect on the time constant of recovery from inactivation. To study the molecular determinants of Kv4.3, we performed alanine scanning on a conserved motif of Kv4.3 and assayed the affinity between mutants and JZTX-V. The results not only showed that I273, L275, V283, and F287 were molecular determinants in the conserved motif of Kv4.3 for interacting with JZTX-V but also speculated the underlying action mechanism that the hydrophobic interaction and steric effects played key roles in the binding of JZTX-V with Kv4.3. In summary, our studies have laid a scientific theoretical foundation for further research on the interaction mechanism between JZTX-V and Kv4.3.

Kinetic and Molecular Docking Studies to Determine the Effect of Inhibitors on the Activity and Structure of Fused G6PD::6PGL Protein from Trichomonas vaginalis.

Trichomoniasis is a sexually transmitted disease with a high incidence worldwide, affecting 270 million people. Despite the existence of a catalog of available drugs to combat this infection, their extensive use promotes the appearance of resistant Trichomonas vaginalis (T. vaginalis), and some side effects in treated people, which are reasons why it is necessary to find new alternatives to combat this infection. In this study, we investigated the impact of an in-house library comprising 55 compounds on the activity of the fused T. vaginalis G6PD::6PGL (TvG6PD::6PGL) protein, a protein mediating the first reaction step of the pentose phosphate pathway (PPP), a crucial pathway involved in the parasite’s energy production. We found four compounds: JMM-3, CNZ-3, CNZ-17, and MCC-7, which inhibited the TvG6PD::6PGL protein by more than 50%. Furthermore, we determined the IC50, the inactivation constants, and the type of inhibition. Our results showed that these inhibitors induced catalytic function loss of the TvG6PD::6PGL enzyme by altering its secondary and tertiary structures. Finally, molecular docking was performed for the best inhibitors, JMM-3 and MCC-7. All our findings demonstrate the potential role of these selected hit compounds as TvG6PD::6PGL enzyme selective inhibitors.

Inhibition of cytochrome P450 enzymes and uridine 5′-diphospho-glucuronosyltransferases by vicagrel in human liver microsomes: A prediction of potential drug-drug interactions

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 μM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4′-hydroxylation with Ki values of 0.19 μM and 1.2 μM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min−1 and 1.52 μM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 μM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin β-D-glucuronidation with a Ki value of 5.6 μM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.

Evaluation of Inactivation Kinetics and Thermodynamic Properties of Peroxidase from Cucumis sativus for Biotechnological and Industrial Applications

The unpurified enzyme gave specific activity of 51.36 μ/mg and thereafter it was subjected to two phases of purification process of salt precipitation and gel filtration. The precipitating agent was ammonium sulphate while Sephadex-G100 served as purification matrix. The purification fold achieved after precipitation and filtration was 3.3 and with corresponding specific activities of 34.22 μ/mg and 116.31 μ/mg. The substrate used for the assay was o-dianisidine. Within 40-80°C of temperature, the kinetics of the peroxidase inactivation was evaluated. The results from assays showed that cucumber peroxidase conformed to the hypothesis of Michealis-Menten Theory. From the Lineweaver-Burk plot, Michaelis Constant (Km) and maximum velocity (Vmax) were evaluated and obtained 5.02mg/ml and 11.57μmol/min respectively. The heat induced inactivation gave biphasic curves, where initial rise in temperature was rapidly succeeded with much slower decrease. A first-order kinetic behaviour was observed for cucumber peroxidase heat inactivation. The k values of between 3.49×10-2 to 8.38×10-2 min-1 was obtained while the Z value was found to be 22.3°C. Decrease in k values with rise in temperature suggests that cucumber peroxidase was rapidly inactivated at elevated temperature. The slope of Arrhenius plot gave the activation energy of 127.99KJMol-1K-1. Also evaluated were thermodynamic constants (ΔH, G Δ, ΔS) for inactivation of peroxidase at variable temperatures. Cucumber peroxidase activity was observed to be pH sensitive and stable within pH range of 5.6-8. Further decrease or increase from this range resulted to decrease in peroxidase stability.

Synergistic algicidal effects of combined UV-LED/chlorine treatments on Tetraselmis sp.: Optimization and mode-of-action

• Disinfection of Tetraselmis sp. using UV-LEDs, chlorine and UV-LED/chlorine. • UV-LED/chlorine promotes algal inactivation compared to individual treatments. • UV-LED/chlorine disinfection is a wavelength-dependent process. • UV 285 /chlorine showed the best inactivation effect and the lowest energy-efficiency. • Radicals were found to play an important role in the synergy treatment. Unwanted proliferation of and adhesion by microalgae, as involved in fouling and species invasion, are important problems across many maritime operations and coastal industrial activities. The algicidal mechanisms of ultraviolet light-emitting diodes (UV-LEDs) and the synergistic algicidal effects between UV-LEDs and chlorine treatments in seawater were investigated in this study. Tetraselmis sp. was employed as the challenge organism. Five UV wavelengths (265, 280, 285, 300 and 315 nm) and three chlorine dosages (0.6, 0.8 and 1 mg L −1 ) were examined in a factorial experiment to optimize the disinfection performance achieved by combined UV-LED/chlorine treatments in seawater. Overall, the combined treatments showed a more effective inactivation of the microalgae than did the single treatments. Treatment with UV 285 combined with 0.8 mg L −1 chlorine resulted in 3.12-log inactivation at 100 mJ cm −2 (i.e. 2.34 Einsteins L -1 , 67 s). This optimal combination showed a higher inactivation rate constant and lower energy consumption (E E,2 ) than did the other treatments. The contribution of UV-LEDs, chlorine and hydroxyl radical (·OH) in the inactivation of Tetraselmis sp. by the combined UV-LED/chlorine disinfection process was investigated to illustrate the mechanism of this synergistic process, the results indicated that ·OH generated by photolysis were not the only contributors to the inactivation of the microalgae. Although a UV 300 /chlorine treatment demonstrated the highest chlorine photolysis rate, the inactivation constant of this treatment was lower than that of UV 285 /chlorine. These results demonstrated a synergistic algicidal effect of the combined disinfection system caused by exposure to the 285 nm UV-LED and low dosages of chlorine, which could provide an important basis for further developments and possibly industrial applications in the future.

Production and Characterization of Whole-Cell Rhizopus oryzae CCT3759 to be Applied as Biocatalyst in Vegetable Oils Hydrolysis

The present study aims to produce a mycelium-bound lipase of the fungus Rhizopus oryzae CCT3759 by submerged fermentation in order to be applied as biocatalyst in the hydrolysis of different vegetable oils. Optimal cultivation conditions have been achieved in a medium containing olive oil as inducer for 72 h of fermentation, thus obtaining 30.5 g/L of dry biomass concentration and hydrolytic activity of 389.1 U/g, which corresponds to a total lipase activity around of 12,000 U/L. Maximum hydrolytic activity was observed at pH 6.0 and 40 °C. Kinetic parameters concerning apparent Michaelis–Menten constant (Km = 50.5 mM) and maximum reaction rate (Vmax = 815.4 µmol/g min) have been determined in olive oil emulsion hydrolysis. Thermal stability tests revealed that the enzyme retained 75% of its initial activity after 4 h at 50 °C, whose thermal inactivation constant (Kd) and half-life (t1/2) was 0.073 h−1 and 9.4 h, respectively. The effect of biocatalyst concentration, expressed as activity units—U (200 and 400 U), on the hydrolysis of vegetable oils was investigated under fixed conditions: oil/buffer mass ratio of 25% (m/m), 100 mM buffer sodium phosphate pH 6.0, 40 °C and the mechanical stirring frequency of 600 rpm. As expected, increasing the initial activity from 200 to 400 U leads to higher values of initial reaction rates and hydrolysis percentage. However, initial reaction rate values were similar for six different vegetable oils due to the high accessibility of the lipase to the substrate under such experimental conditions. A complete hydrolysis of olive, cottonseed, sunflower and canola oils has been achieved after 26–30 h of reaction using 400 U of activity. These results suggest a promising application of the produced biocatalyst in the production of free fatty acids, an important class of compounds for oleochemical industries.

Preparation of cross-linked enzyme aggregates of lipase from Aspergillus niger: process optimization, characterization, stability, and application for epoxidation of lemongrass oil.

Cross-linked enzyme aggregates (CLEAs) of lipase were prepared after fractional precipitation with 40-50% ammonium sulfate and then cross-linking with glutaraldehyde. The process variables for the preparation of lipase-CLEAs such as glutaraldehyde concentration, cross-linking period, and initial pH of medium were optimized. The optimized conditions for the preparation of lipase-CLEAs were 25mM/80min/pH 7.0, and 31.62mM/90min/pH 6.0 with one factor at a time approach and numerical optimization with central composite design, respectively. Lipase-CLEAs were characterized by particle size analysis, SEM, and FTIR. Cross-linking not only shifted the optimal pH and temperature from 7.0 to 7.5 and 40-45 to 45-50°C, but also altered the secondary structure. Lipase-CLEAs showed an increase in Km by 7.70%,and a decrease in Vmax by 16.63%. Lipase-CLEAs presented better thermostability than free lipase as evident from thermal inactivation constants (t1/2, D and Ed value), and thermodynamic parameters (Ed, ΔH°, ΔG°, and ΔS°) in the range of 50-70°C. Lipase-CLEAs retained more than 65% activity up to four cycles and showed good storage stability for 12days when stored at 4 ± 2°C. They were successfully utilized for the epoxidation of lemongrass oil which was confirmed by changes in iodine value, epoxide value, and FTIR spectra.

Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism.

BackgroundVentricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α2-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated.MethodsA conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (If), potassium (K+) channel currents (IK1 and IKr), sodium (Na+) channel currents (INa), and calcium (Ca2+) channel currents (ICa) in ventricular-like hiPSC-CMs.ResultsDMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC50) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD90). DMED also inhibited the amplitudes of the INa and ICa without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na+ and Ca2+ channel activation at potential –40 to –20 mv, and –20 mv. DMED increased the time constant of inactivation of the Na+ and Ca2+ channels. However, DMED did not affect the IK1, IKr, If, and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na+ and Ca2+ channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine.ConclusionsDMED could inhibit the frequency of spontaneous action potentials and decrease the INa and ICa of hiPSC-CMs via mechanisms that were independent of the α2-adrenoceptor, the imidazoline receptor, and the α1-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

Block of Voltage-Gated Sodium Channels by Atomoxetine in a State- and Use-dependent Manner.

Atomoxetine, a neuroactive drug, is approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). It is primarily known as a high affinity blocker of the noradrenaline transporter, whereby its application leads to an increased level of the corresponding neurotransmitter in different brain regions. However, the concentrations used to obtain clinical effects are much higher than those which are required to block the transporter system. Thus, off-target effects are likely to occur. In this way, we previously identified atomoxetine as blocker of NMDA receptors. As many psychotropic drugs give rise to sudden death of cardiac origin, we now tested the hypothesis whether atomoxetine also interacts with voltage-gated sodium channels of heart muscle type in clinically relevant concentrations. Electrophysiological experiments were performed by means of the patch-clamp technique at human heart muscle sodium channels (hNav1.5) heterogeneously expressed in human embryonic kidney cells. Atomoxetine inhibited sodium channels in a state- and use-dependent manner. Atomoxetine had only a weak affinity for the resting state of the hNav1.5 (Kr: ∼ 120 µM). The efficacy of atomoxetine strongly increased with membrane depolarization, indicating that the inactivated state is an important target. A hallmark of this drug was its slow interaction. By use of different experimental settings, we concluded that the interaction occurs with the slow inactivated state as well as by slow kinetics with the fast-inactivated state. Half-maximal effective concentrations (2–3 µM) were well within the concentration range found in plasma of treated patients. Atomoxetine also interacted with the open channel. However, the interaction was not fast enough to accelerate the time constant of fast inactivation. Nevertheless, when using the inactivation-deficient hNav1.5_I408W_L409C_A410W mutant, we found that the persistent late current was blocked half maximal at about 3 µM atomoxetine. The interaction most probably occurred via the local anesthetic binding site. Atomoxetine inhibited sodium channels at a similar concentration as it is used for the treatment of ADHD. Due to its slow interaction and by inhibiting the late current, it potentially exerts antiarrhythmic properties.

The effects of tegaserod, a gastrokinetic agent, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells.

Tegaserod, a gastroprokinetic agent, is used to treat irritable bowel syndrome. Despite its extensive clinical use, little is known about the effects of tegaserod on vascular ion channels, especially K+ channels. Therefore, we examined the effects of tegaserod on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Tegaserod inhibited Kv channels in a concentration-dependent manner with an IC50 value of 1.26±0.31µmol/L and Hill coefficient of 0.81±0.10. Although tegaserod had no effect on the steady-state activation curves of the Kv channels, the steady-state inactivation curve was shifted toward a more negative potential. These results suggest that tegaserod inhibits Kv channels by influencing their voltage sensors. The recovery time constant of channel inactivation was extended in the presence of tegaserod. Furthermore, application of train steps (1 and 2Hz) in the presence of tegaserod progressively increased the inhibition of Kv currents suggesting that tegaserod-induced Kv channel inhibition is use (state)-dependent. Pretreatment with a Kv1.5 subtype inhibitor suppressed the Kv current. However, additional application of tegaserod did not induce further inhibition. Pretreatment with a Kv2.1 or Kv7 inhibitor did not affect the inhibitory effect of tegaserod on Kv channels. Based on these results, we conclude that tegaserod inhibits vascular Kv channels in a concentration- and use (state)-dependent manner independent of its own functions. Furthermore, the major Kv channel target of tegaserod is the Kv1.5 subtype.

Involvement of pore helix in voltage-dependent inactivation of TRPM5 channel

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent-permeable cation channel that is activated by intracellular Ca2+. Expression of TRPM5 has been shown in taste cells, pancreas, brainstem and olfactory epithelium, and this channel is thought to be involved in controlling membrane potentials. In whole-cell patch-clamp recordings, TRPM5 exhibited voltage-dependent inactivation at negative membrane potentials and time constant of voltage-dependent inactivation of TRPM5 did not depend on the intracellular Ca2+ concentrations between 100 and 500 nM. Alanine substitution at Y913 and I916 in the pore helix of TRPM5 increased time constant of voltage-dependent inactivation. Meanwhile, voltage-dependent inactivation was reduced in TRPM5 mutants having glycine substitution at L901, Y913, Q915 and I916 in the pore helix. From these results, we conclude that the pore helix in the outer pore loop might play a role in voltage-dependent inactivation of TRPM5.

Kinetic model for the enzymolysis of Paphia undulate by marine low-temperature alkaline proteinase

The kinetics of enzymatic hydrolysis of paphia sinensis was studied. The molecular weight distribution of the small peptide mixture was determined by liquid and mass spectrometry. The effects of initial substrate concentration on enzymatic hydrolysis reaction, the effects of initial enzyme concentration on enzymatic hydrolysis reaction, the effects of enzymatic hydrolysis time on enzymatic hydrolysis reaction and the mechanism of enzymatic hydrolysis reaction were investigated. In this experiment, the basic kinetic model of enzymatic hydrolysis of protein was derived, and the kinetic parameters and enzyme inactivation constant were determined.