Zheng Fan

Interrelation between pinacidil and intracellular ATP concentrations on activation of the ATP-sensitive K+ current in guinea pig ventricular myocytes.

The patch-clamp technique was used to study the relation between pinacidil and intracellular ATP concentration [( ATP]i) on the activation of the outward K+ current in guinea pig ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 microM or higher concentrations activated the time-independent outward current at potentials positive to -80 mV, and the pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression of the maximum response at increased [ATP]i. The pinacidil-induced shortening of the action potential duration and outward current were inhibited by application of 0.3-1.0 microM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive K+ channel current without changing the unitary amplitude, and increased open probability of the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was blocked by glibenclamide. These results prove the notion that pinacidil activates the ATP-sensitive K+ channel current, which explains the action potential shortening in cardiac cells after application of pinacidil.

Pinacidil activates the ATP-sensitive K+ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of pinacidil on the adenosine-5′-triphosphate (ATP)-sensitive K+ channel current in guinea-pig ventricular myocytes. In inside-out patches, the ATP-sensitive K+ channel current could be recorded at an internal ATP concentration of 0.5 mM or less and almost complete inhibition was achieved by raising the concentration to 2 mM. Application of pinacidil (10–30 μM) in the presence of 2 mM ATP restored the current, whereas 5 mM ATP antagonized the effect of pinacidil. The conductance of the channel at symmetrical K+ concentrations of 140 mM was 75 pS with a slight inward rectification at voltages positive to +40 mV. There was no significant change in the conductance after application of pinacidil. In 0.5 mM ATP, at −80 mV, both the distributions of the open time and the life-time of bursts could be fitted by a single exponential. An increase in ATP concentration decreased the mean life-time of bursts, whereas pinacidil increased it with little increase in the mean open time. Closed time distributions of the channel were fitted by at least two exponentials, with a fast and a slow time constant. An increase in ATP concentration markedly increased the slow time constant associated with a decrease in the number of bursts, whereas the effect of pinacidil was opposite to that of increased ATP. These results indicate that pinacidil increases the open-state probability of the ATP-sensitive K+ channel. In cell-attached patches, application of pinacidil (100–200 μM) to the extracellular solution reversibly induced the channel activity, which showed similar properties to those of the ATP-sensitive K+ channel recorded in cell-free patches.

Two components of use-dependent block of Na+ current by disopyramide and lidocaine in guinea pig ventricular myocytes.

We studied the kinetics of the use-dependent block of the Na+ current (INa) by disopyramide and lidocaine. INa was recorded from isolated guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The use-dependent block of INa by disopyramide with 20- and 200-msec depolarizing pulses developed in two exponential functions. The degree of the use-dependent block and the amplitude of the fast (Af) and slow (As) components with the short (20-msec) pulse protocol were comparable to those with the long (200-msec) pulse protocol. When pH was raised from 7.3 to 8.0, disopyramide increased Af without a change in As. At pH 6.5, INa block developed with a single exponential function revealing only the slow component. The fast and slow components of INa block by disopyramide could be explained by binding of the uncharged and charged forms, respectively, to the activated state of the channel. Development of INa block by lidocaine also was expressed by two exponentials at all pulse durations (5-200 msec). As pulse durations were prolonged or holding potentials were depolarized, the degree of the use-dependent block and Af increased. When pH was lowered to 6.5, the short pulse produced only the slow component, whereas the long pulse caused two exponentials with decreased Af and increased As. Internal application of QX-314, a permanently charged lidocaine analogue, produced a single exponential block of INa with a very slow onset rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of nicorandil on ATP‐sensitive K+ channel in guinea‐pig ventricular myocytes

1 Patch‐clamp techniques were used to study the effects of nicorandil (2‐nicotinamiodethyl nitrate) on the adenosine 5′‐triphosphate (ATP)‐sensitive K+ channel current (IK.ATP) in guinea‐pig ventricular myocytes. 2 Nicorandil activated the time‐independent outward current. This effect was dependent on intracellular ATP concentration ([ATP]i) showing a larger effect at 2 mm than at 10 mm [ATP]i The nicorandil‐induced outward current was inhibited by application of 0.3 μm glibenclamide. 3 In the inside‐out patch configuration, 0.3–1.0 mm nicorandil increased the open‐state probability of IK.ATP without a change in its conductance value (about 90pS). This effect was inhibited by glibenclamide. Analysis of the open and closed time distributions showed that nicorandil had no effect on open and closed distributions shorter than 5 ms. On the other hand, nicorandil increased the life time of bursts and decreased the interburst intervals. 4 The inward rectifier K+ channel current was not influenced by internal application of nicorandil. 5 Therefore, we conclude that IK.ATP is the only K+ current activated by nicorandil, and the main effect of nicorandil is on the kinetics of the IK.ATP bursting behaviour. These actions are similar to that of pinacidil on this preparation.

Use-dependent block of Na+ currents by mexiletine at the single channel level in guinea-pig ventricular myocytes.

1 The mechanism of use‐dependent block of Na+ current by mexiletine was studied at the single channel level in guinea‐pig ventricular myocytes by the patch‐clamp techniques. All experiments were performed using stimulation protocols to enable us to analyze the strict dependence of changes in channel properties on channel use. 2 In cell‐attached patches, bath or pipette application of mexiletine (40 μm) produced a use‐dependent reduction of the peak average current without changes in single channel conductance. Null sweeps were increased and the number of openings per sweep decreased with successive pulses, whereas no significant change in the mean open time was detected during the train. 3 Block by mexiletine became greater when pulse duration was extended beyond the period in which channels were open, suggesting that block progressed without channel opening. 4 At near threshold potentials, mexiletine decreased the later occurrence of first openings. Additionally, late openings were reduced in a use‐dependent way. 5 We conclude that mexiltine binds to the inactivated closed states of the Na+ channel and then causes a failure of late openings as well as early, which results in null sweeps on subsequent depolarization.

The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na+ channel currents. Single Na+ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na+ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na+ channel is directly modulated by phosphorylation process through A-kinase.

Multiple ionic mechanisms of early afterdepolarizations in isolated ventricular myocytes from guinea-pig hearts.

Ionic mechanisms of early afterdepolarization (EAD) induced by the K(+)-free solution or veratridine were studied with guinea-pig ventricular myocytes using the patch-clamp technique of whole-cell and cell-attached patch configurations. In the K(+)-free solution, myocytes exhibited prolonged action potential duration with humps on the final repolarization phase, which eventually turned into EAD starting around -70 mV and induced triggered activity. Application of 0.5 mM Cd2+ inhibited the development of EAD and caused depolarization of maximum diastolic potentials around -30 mV, although Cd2+ did not prevent prolongation of the action potential. Application of 50-100 microM Ni2+ or 30 microM tetrodotoxin had little effects on EAD and diastolic potentials. The background current-voltage relation examined by a ramp voltage clamp showed inhibition of the inward rectifier K+ current, induction of steady inward current between -40 and -10 mV, and increase in the outward tail current upon repolarization in the K(+)-free solution. Cd2+ completely blocked the steady inward current at the plateau level and partially depressed the delayed outward K+ current, while Ni2+ had no effects on the background I-V relation. Tetrodotoxin showed a mild inhibitory effect on the inward component of the background current negative to -50 mV, but left the steady inward current at the plateau level. Therefore, EAD in the K(+)-free condition is mainly formed by decreased inward rectifier K+ current, activation of the L-type Ca2+ current, and time-dependent decay of the delayed outward K+ current upon repolarization. Application of 25-100 microM veratridine caused marked prolongation of action potential with appearance of regenerative EADs. Action potential prolongation and EADs were partially abolished by Cd2+ and completely eliminated by tetrodotoxin. The single channel current recordings showed a decreased current amplitude, and prolonged and delayed openings of the Na+ channel currents by veratridine. Thus, an ensemble average current showed markedly prolonged decay time constant of 609 msec in veratridine from 3.6 msec in the control. These results indicate that veratridine-induced EAD is mainly formed by altered properties of the Na+ channel current and partly by the L-type Ca2+ current due to slowed repolarization. Thus, EAD can be induced by different ionic mechanisms depending on the basal conditions.

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes.

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes.

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxy-benzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxy-acetophenone, on the adenosine-5′-triphosphate(ATP)-sensitive K+ channel current (IK.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5–1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of IK.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K+ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as IK.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on IK.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of IK.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate IK.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c) may require an ADP-dependent process.

Amantadine-induced afterpotentials and automaticity in guinea pig ventricular myocytes.

The ionic mechanisms of amantadine-induced changes in membrane potential and automatic activity in guinea pig ventricular myocytes were studied using the suction-pipette whole-cell clamp method. While 25-100 μM amantadine decreased the action potential amplitude and duration, 200 and 400 μM amantadine lengthened the action potential duration and decreased the maximum diastolic potential with an appearance of diastolic depolarization and automaticity. In the presence of 25-100 μM amantadine, the preparations developed an afterpotential due to incomplete repolarization and a delayed afterdepolarization that eventually brought about triggered automaticity. The former type of afterpotential was abolished by tetrodotoxln (TTX) and the latter by Co2+. Spontaneous activity from the diastolic depolarization was also abolished by Co2+ but not by Cs+. Amantadine suppressed the calcium current to as much as half of the control at the concentrations used (25-200 μM). The drug also produced a depression of the inward rectifier K+ current. The outward current showing time-dependent decay was activated at the plateau voltages by concentrations lower than 100 μM, whereas the delayed outward K+ current was depressed by the drug in a concentration-dependent manner at more positive potentials. Amantadine activated the TTX-sensitive and TTX-insensitive inward currents on repolarization from depolarized states, without producing the transient inward current. These results indicate that the amantadine-induced diastolic depolarization and afterpotentials are caused by changes in multiple ionic currents and that, therefore, the drug can be used as a unique model for the study of arrhythmogenesis.