Tetrahydroacridine inhibits voltage-dependent Na^＋ current in guinea-pig ventricular myocytes

AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na^ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na^ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na^ current with an IC50 value of 120μmol/L(95% confidence range: 108-133μmol/L). (2) The inhibitory effects of tacrine on Na^ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100μmol/L caused a negative shift (about 10mV) in the voltage-dependence of steady-state inactivation of Na^ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na^ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na^ channels in guinea-pig ventricular myocytes. Tacrine acted as inactivation stabilizer of Na^ channels in cardiac tissues.     

Inhibitory effects of tetrandrine on the Na^＋ channel of human atrial fibrillation myocardium

Aim： Tetrandrine （Tet） is a Ca^2＋ channel blocker and has antiarrhythmic effects. Less information exists with regard to the mechanisms underlying its antiarrhythmic action other than blocking Ca^2＋ channels. In this study, the effects of Tet on the Na^＋ current （INa） in the atrial myocardium of patients in atrial fibrillation （AF） and sinus rhythm （SR） were investigated, and the characteristics of the Na^＋ current were synchronously compared between the AF and SR patients. Methods： Na^＋ currents were recorded using the whole-cell patch clamp technique in single atrial myocyte of the AF and the normal SR groups. The effects of Tet （40-120μmol/L） on the Na^＋ current in the two groups were then observed. Results： Tet （60-120 pmol/L） decreased INa density in a concentration-dependent manner and made the voltage-dependent activation curve shift to more positive voltages in the SR and AF groups. After exposure to Tet, the voltage-dependent inactivation curve of INa was shifted to more negative voltages in the two groups. Tet delayed the time-dependent recovery of INa in a concentration dependent manner in both AF and SR cells; however, there were no differences in the effects of Tet on INa density and properties in the two groups. The INa density of AF patients did not differ from that of the SR patients. Conclusion： Tet can block sodium channels with slow recovery kinetics, which may explain the mechanisms underlying the antiarrhythmic action of Tet. The decreased conduction velocity （CV） in AF patients is not caused by the Na^＋ current.     

Effects of SKF83959 on the excitability of hippocampal CA1 pyramidal neurons： a modeling study

Aim： 3-Methyl-6-chloro-7,8-hydroxy-1-（3-methylphenyl）-2,3,4,5-tetrahydro-1H-3-benzazepine （SKF83959） have been shown to affect several types of voltage-dependent channels in hippocampal pyramidal neurons. The aim of this study was to determine how modulation of a individual type of the channels by SKF83959 contributes to the overall excitability of CA1 pyramidal neurons during either direct current injections or synaptic activation.
Methods： Rat hippocampal slices were prepared. The kinetics of voltage-dependent Na^＋ channels and neuronal excitability and depolarization block in CA1 pyramidal neurons were examined using whole-cell recording. A realistic mathematical model of hippocampal CA1 pyramidal neuron was used to simulate the effects of SKF83959 on neuronal excitability.
Results： SKF83959 （50 μmol/L） shifted the inactivation curve of Na^＋ current by 10.3 mV but had no effect on the activation curve in CA1 pyramidal neurons. The effects of SKF83959 on passive membrane properties, including a decreased input resistance and depolarized resting potential, predicted by our simulations were in agreement with the experimental data. The simulations showed that decreased excitability of the soma by SKF83959 （examined with current injection at the soma） was only observed when the membrane potential was compensated to the control levels, whereas the decreased dendritic excitability （examined with current injection at the dendrite） was found even without membrane potential compensation, which led to a decreased number of action potentials initiated at the soma. Moreover, SKF83959 significantly facilitated depolarization block in CA1 pyramidal neurons. SKF83959 decreased EPSP temporal summation and, of physiologically greater relevance, the synaptic-driven firing frequency.
Conclusion： SKF83959 decreased the excitability of CA1 pyramidal neurons even though the drug caused the membrane potential depolarization. The results may reveal a partial mechanism for the drug’s anti-Parkinsonian effects and may also suggest that SKF83959 has a potential antiepileptic effect.     

Effects of docosahexaenoic acid on large-conductance Ca^2＋-activated K^＋ channels and voltage-dependent K^＋ channels in rat coronary artery smooth muscle cells

Aim： To investigate the effects of docosahexaenoic acid （DHA） on large-conductance Ca^2＋-activated K^＋ （BKca） channels and voltage-dependent K^＋ （Kv） channels in rat coronary artery smooth muscle cells （CASMCs）.
Methods： Rat CASMCs were isolated by an enzyme digestion method. BKCa and Kv currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BKCa and Kv channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L. Results： When DHA concentrations were greater than 10 μmol/L, BKCa currents increased in a dose-dependent manner. At a testing potential of ＋80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BKCr currents were increased at the above concentrations, respectively. The half-effective concentration （EC50） of DHA on BKca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, Kv currents were gradually blocked by increasing concentrations of DHA. At a testing potential of ＋50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of Kv currents were blocked at the different concentrations listed above, respectively. The EC50 of DHA on Kv currents was 44.20±0.63 μmol/L.
Conclusions： DHA can activate BKca channels and block Kv channels in rat CASMCs, and the EC50 of DHA for BKca channels is lower than that for Kv channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BKCa channels.     

Biphasic effects of haloperidol on sodium currents in guinea pig ventricular myocytes

Aim： To study the effects of haloperidol on sodium currents （INa） in guinea pig ventricular myocytes. Method： Whole-cell patch clamp technique was employed to evaluate the effects of haloperidol on INa in individual ventricular myocytes. Results： Haloperidol （0.1-3 wnol/L） inhibited INa in a concentration-dependent manner with an IC50 of 0.253±0.015 larnol/L. The inhibition rate of haloperidol （0.3 μmol/L） on INa was 22.14%±0.02%, and the maximum conductance was reduced. Haloperidol significantly reduced the midpoints for the activation and inactivation of INa by 2.09 and 4.09 mV, respectively. The time constant of recovery was increased. The increase in time intervals could only recover by 90.14%±1.4% （n=6）; however, haloperidol at 0.03 μmol/L enhanced INa conductance. The midpoints for the activation and inactivation Of INa were shifted by 1.38 and 5.69 mV, respectively, at this concentration of haloperidol. Conclusion： Haloperidol displayed a biphasic effect on INa in guinea pig cardiac myocytes. High concentrations of haloperidol inhibited INa, while lower concentrations of haloperidol shifted the activation and inactivation curve to the left. Full recovery of recovery curve was not achieved after 0.3 μmol/L haloperidol administration, indicating that the drug affects the inactivated state of sodium channels.     

Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

Aim： KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopus laevis oocytes. Methods： Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes. Results： Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K~ current was almost completely blocked by a KCNQ4 blocker, linopirdine （0.25 mmol/L）. BIMI （a PKC inhibitor） prevented the effects of PMA （a PKC activator） on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine （2 pmol/L）, calyculin A （2 pmol/L） or okadaic acid （1 pmol/L）, caused a significant positive shift in V1/2 and a decrease in the conductance of KCNQ4 channels. The V1/2 was shifted from -14.6±0.5 to -6.4±0.4 mV by cyclosporine, -18.8±0.5 to -9.2_＋0.4 mV by calyculin A, and -14.1±0.5 to -0.7＋0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors （okadaic acid or calyculin A） on the induction of a positive shift of V1/2 were augmented by further addition of PMA. Conclusion： These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.     

Mechanism of regulation of CaCC by the Mas-related G protein-coupled receptor D

MrgD is expressed almost exclusively in dorsal root ganglion(DRG) neurons.And its activation inhibited KCNQ/M-currents that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.Ca2+-activated chloride channels(CaCCs) are found in DRG neurons and regulate neuronal cell excitability as well.But the interaction between CaCCS and MrgD is still unknown.We here found that β-alanine-induced activation of MrgD resulted in eliciting Ca2+-activated chloride currents.The currents were inhibited by flufenamic acid(FFA) and by inhibition of phospholipase C and Ca2+ chelating agent EGTA.However,calphostin C,a PKC inhibitor,had no effect on the currents.These present data show that the inward currents induced by activation of MrgD were mediated through Gq-phospholipase C-IP3-Ca2+ release pathway,but not via Gi pathway.     

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain

Aim： The objectives of this study were to investigate the inhibitory action of verapamil on wild-type（WT） and mutation HERG K^＋ channel current （IHERG）, and to determine whether mutations in the S6 region ale important for the inhibition of IHERG by verapamil. Methods： HERG channels （WT, Y652A, and F656A） were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage-clamp technique. Results： WT HERG is blocked in a concentration-dependent manner by verapamil （half-maximal inhibition concentration [IC50]=5.1 μmol/L）, and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC50 for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers. Conclusion： Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner （S6） helix, are critical in the verapamil-binding site.     

Blockade of paeoniflorin on sodium current in mouse hippocampal CA_1 neurons

AIM: To study the blockade of paeoniflorin (Pae) on I_(Na) in the acutely isolated hippocampus neurons of mice. METHODS: The whole-cell patch clamp technique was used. RESULTS: Pae inhibited I_(Na) in frequency-dependent and concentration-dependent manners, with an IC_(50) of 271μmol/L. Pae 0.3 mmol/L shifted the activation potential of the maximal I_(Na) from -40 mV to -30 mV, shifted the steady-state activation and inactivation curves toward more positive and negative potentials by 10.8 mV, and 18.2 mV, respectively, and postponed the recovery of I_(Na) inactivation state from (4.2±0.7) ms to (9.8±1.2) ms. CONCLUSION: Pae inhibited I_(Na) in mouse hippocampus neurons.     

Inhibition of 5-HT3 receptor cation channels by ifenprodil in excised patches of N1E-115 cells

The patch-clamp technique was applied in outside-out patches of N1E-115 mouse neuroblastoma cells to investigate the effects of ifenprodil [(±) erythreo-ifenprodil tartrate], a drug with neuroprotective properties in cerebral ischemia, on the inward currents through 5-HT₃ receptor channels. A high time resolution was achieved by using a rapid solution exchange system (exchange rate <1 ms). Ifenprodil inhibited the peak currents evoked by 30 μM 5-HT in a concentration-dependent but voltage-independent manner. The effect was most potent when ifenprodil was continuously applied to the patches 45 s before and during the 2-s administration of 5-HT (IC₅₀=16 μM) and it was only slightly less potent when it was applied during the 45 s prior to 5-HT only (IC₅₀=29 μM). When applied in this manner, ifenprodil also produced a concentration-dependent increase of the onset time constant (τ_ON) of the 5-HT (30 μM)-induced currents. When the drug was exclusively co-applied with 5-HT, ifenprodil was least potent in inhibiting the peak currents (IC₅₀=98 μM), and it had no effect on the current onset kinetics. All protocols of ifenprodil application accelerated current inactivation as reflected by a decrease of the current inactivation time constant (τ_OFF). All effects of ifenprodil were reversible after washout periods of 2–5 min. In conclusion, the potency of ifenprodil in inhibiting the inward current through 5-HT₃ receptor channels is strongly dependent on the application protocol: presence of the drug before the agonist-induced activation of the 5-HT₃ receptor channels is necessary for a relatively potent inhibition of the 5-HT-induced peak current and is a prerequisite for the prolongation of τ_ON; in addition, a weak but fast inhibitory effect on the current amplitude and decay constant of the 5-HT-induced current was revealed by the experiments in which ifenprodil was exclusively present during exposure to 5-HT. Three alternatives compatible with the components of the ifenprodil effect have been discussed: (1) different effects of the two enantiomers, (2) action via two different mechanisms, and (3) operation via a single mechanism only. Received: 9 October 1997 / Accepted: 7 May 1998     

Puerarin blocks Na~+ current in rat ventricular myocytes

AIM: To study the effect of puerarin (Pue) on Na~+ channel in rat ventricular myocytes. METHODS: Whole-cell patch-clamp technique was applied on isolated cardiomyocytes from rats. RESULTS: Pue inhibited cardiac I_(Na) in a positive rate-dependent and dose-dependent manner, with an IC_(50) of 349 μmol/L. The kinetics of blockage of cardiac sodium channel by Pue resembled the ClassIa/Ic of antiarrhythmic agents. Pue 300 μmol/L did not alter the shape of the I-V curve of I_(Na), but markedly shifted the steady-state inactivation curve of I_(Na) towards more negative potential by 15.9 mV, and postponed the recovery of I_(Na) inactivation state from (21.9±1.6) ms to (54.4±3.4) ms (P<0.01 ). It demonstrated that the steady state of inactivation was affected by Pue significantly. CONCLUSION: Pue protected ventricular myocytes against cardiac damage and arrhythmias by inhibiting recovery from inactivation of cardiac Na~+ channels.     

Genistein inhibits carotid sinus baroreceptor activity in anesthetized male rats

Aim: To study the effect of genistein (GST) on carotid baroreceptor activity (CBA). Methods: The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. Results: GST at 50, 100, and 200 μmol/L inhibited the CBA, which shifted FCCB to the right and downward, with a marked decrease in peak slope and peak integral value of carotid sinus nerve discharge in a concentration-dependent manner. Pretreatment with 100 μmol/L N^G-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not affect the effect of GST on CBA. Pretreatment with 500 nmol/L Bay K8644, an agonist of calcium channels, could completely abolish the effect of GST on CBA. A potent inhibitor of tyrosine phosphatase, sodium orthovanadate (1 mmol/L), could attenuate the inhibitory effect of GST. Conclusion: GST inhibits CBA, and the effect may be mediated by protein tyrosine kinase inhibition and a decrease in Ca^2 influx through the stretchactivated channels.     

Involvement of cAMP/cAMP-dependent protein kinase signaling pathway in regulation of Na^＋, K^＋-ATPase upon activation of opioid receptors by morphine

The depolarization of neurons induced by impairment of Na^＋ , K^＋ - ATPase activity after chronic opiate treatment has been shown to involve the development of opioid dependence. However, the mechanisms underlying changes in Na^＋ , K^＋ - ATPase activity following opioid treatment are unclear. The best - established molecular adaptation to chronic opioid exposure is up - regulation of the cAMP/PKA signaling pathway, this study, therefore, was undertaken to investigate the role of up - regulation of cAMP/ PKA signaling pathway in alteration of the mouse hippocampal Na^＋, K^＋ - ATPase activity. The results demonstrated that acute morphine treatment dose - dependently stimulated Na^＋ , K^＋ - ATPase activity. This action could be significantly suppressed by adenylyl cyclase activator forskolin, or the cAMP analogue db - cAMP. Contrary to acute morphine treatment, chronic morphine treatment significantly inhibited Na^＋ , K^＋-ATPase activity. Moreover, an additional decrease in Na^＋, K^＋ - ATPase activity was observed by naloxone precipitation. The effects of both acute and chronic morphine treatment on Na^＋, K^＋ -ATPase activity were naltrexone - reversible. The regulation of Na^＋ , K^＋ - ATPase activity by morphine was inversely correlated with intracellular cAMP accumulation. H -89, a specific PKA inhibitor, mimicked the stimulatory effect of acute morphine but antagonized the inhibitory effect of chronic morphine on Na^＋ , K^＋-ATPase activity. However, okadaic acid, a protein phosphatase inhibitor, suppressed acute morphine stimulation but potentiated chronic morphine inhibition of Na^＋, K^＋ - ATPase activity. The regulation of Na^＋ , K^＋ - ATPase activity by morphine treatment appeared to associate with the alteration in phosphorylation level, but not to be relevant to the change in abundance of Na^＋, K^＋ - ATPase.     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

Effects of （-）-stepholidine on NMDA receptors： comparison with haloperidol and clozapine

Aim： To examine whether （-）-stepholidine （SPD） has a direct effect on the N- methyl-D-aspartic acid receptors （NMDAR） containing the NMDA receptor subunits NR2A or NR2B and to compare its effect with those of haloperidol （Hal） and clozapine （Cloz）. Methods： NMDAR was transiently expressed in human embryonic kidney 293 （HEK293） cells. Changes in intracellular calcium concentration （[Ca^2＋]i） induced by NMDAR activation were monitored with Fura-2 ratio imaging techniques. Results： SPD had no significant effects on either subunit of NMDAR at a concentration of less than 100 μmol/L. Hal selectively inhibited NMDAR containing the NR2B subunit, whereas Cloz inhibited both subunits of NMDAR. Although both Hal and Cloz inhibited NRI a/NR2B receptor-mediated Ca^2＋ influx, their effects were different. Hal was more potent and had a faster peak effect than Cloz. Conclusion： Both Hal and Cloz inhibit NMDAR-mediated function, whereas SPD produced only a little inhibition at a high concentration. Based on our other studies, the modulation of SPD on NMDAR function may be via D1 receptor action underlying an indirect mechanism.     

Effect of sugar positions in ginsenosides and their inhibitory potency on Na^＋/K^＋-ATPase activity

Aim： To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi （the roots of Panax ginseng and Panax notoginseng） via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin.
Methods： The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na^＋/K^＋-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na^＋/K^＋-ATPase.
Results： Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na^＋/K^＋-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na^＋/K^＋-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure.
Conclusion： The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na^＋/K^＋-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.     

Acute exposure of methylglyoxal leads to activation of KATP channels expressed in HEK293 cells

Aim： Highly reactive carbonyl methylglyoxal （MGO） is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium （KATP） channel. In the present study we investigated the effects of MGO on the activity of KATP channels.
Methods： Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2Δ36 （a truncated Kir6.2 isoform） alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software.

Results： The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil （10 μmol/L） could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide （10 μmol/L）. MGO （0.1–10 mmol/L） dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Similar results were observed when Kir6.2/SUR2B channels. Kir6.2Δ36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO （3 mmol/L）. Single channel recordings showed that MGO （3 mmol/L） markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered.

Conclusion： Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits.     

The impact of extracellular and intracellular Ca2＋ on ethanol-induced smooth muscle contraction

Aim： To evaluate the impact of extracellular and intracellular Ca2＋ on contractions induced by ethanol in smooth muscle. Methods： Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer. Results： Ethanol （164 mmol/L） produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine （50 and 100 μmol/L）, a local anesthetic agent, and hexamethonium （100 and 500 μmol/L）, a ganglionic blocking agent, failed to affect these contractions, verapamil （1-50 μmol/L） and nifedipine （1-50 pmol/L）, selective blockers of L-type Ca2＋ channels, significantly inhibited the contractile responses of ethanol. Using a Ca2＋-free medium nearly eliminated these contractions in the same tissue. Ryanodine （1-50 μmol/L） and ruthenium red （10-100 pmol/L）, selective blockers of intracellular Ca2＋ channels/ryanodine receptors; cyclopiazonic acid （CPA; 1-10 μmol/L）, a selective inhibitor of sarcoplasmic reticulum （SR） Ca2＋-ATPase; and caffeine （0.5-5 mmol/L）, a depleting agent of intracellular Ca2＋ stores, significantly inhibited the contractile responses induced by ethanol. In addition, the com- bination of caffeine （5 mmol/L） plus CPA （10 μmol/L）, and ryanodine （10 μmol/L） plus CPA （10 μmol/L）, caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine （5 mmol/L）, ryanodine （10 μmol/L） and CPA（IO pmol/L） eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice. Conclusion： Both extracellular and intracellular Ca2＋ may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.     

Role of delayed rectifier K channel currents in β-amyloid （1-40）-induced caspase-3 activation and apoptosis in primary cultured cortical neurons

AIM: To investigate the effect of chronic exposure to/]-amyloid（1-40)(Aβ1-40) on delayed rectifier K^ currents (IK) in primary cultured ratcortical neurons and the effect of IK on caspase-3 activation and neurons apoptosis induced by Aβ1-40. METHODS: K^ currents were recorded using whole-cell patch clamp techniques; cell sviability rate and apoptosis were studied using MTT and Hoechst     

Influence of calcium channel blockers on GABAA receptor

This study was undertaken to address effects of the interactions between calcium transients and serotonergic action on the regulation of sleep architecture and c - Fos expression （as a marker of neuronal activation） in VLPO and TMN induced by pentobarbital （PB） and the influence of Ca^2＋ channel blockers （CCB） on GABAA receptor.