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Benzoylaconine affects on the sodium channel of ventricular
myocytes of Guinea pig
Shu-bin Yang, Hui Sun, Xiao-wei Du, Xi-jun Wang*
National TCM Key Laboratory of Serum Pharmacochemistry, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin, 150040, China.
Abstract: Objective: To investigate the effect of the benzoylaconine on the Na+ current in guinea-pig ventricular myocytes. Material and Methods: The whole cell patch-clamp technique was used to examine the effect of benzoylaconine on fast sodium channel in isolated guinea pig ventricular myocytes. Results:The benzoylaconine (100 uM) increases the magnitude of the peak inward sodium current and Gmax, slow the time course of Na+ channel inactivationD Conclusions: Benzoylaconine modified the gating and kinetics of cardiac Na+ channels in isolated guinea-pig ventricular myocytes.
Benzoylaconine can inscrease the maximum upstrike velocity (Vmax) and prolongs the action potential duration of the cardiac action potential in cultured myocardiocytes of mice(1'2). Action potential prolongation is of particular interest in heart muscle since it represents an antiarrhythythmic principle.It has been known that cardiac excitation is critically dependent on the density and availability of voltage-gated Na channels. availability of voltage-gated Na channels has been assumed to be primarily controlled by voltage. We undertook to further study the effects ofthosemodifying on gating and kinetic fundamental properties by the toxin on Nachannels using whole-cell current recordings, which have been used to examine the effect of a growing number of Na channel toxins in a variety of excitable tissues. Our results show that both conductance and voltage-dependent availability curves are significantly modulated and shifted the activation curves of INa to hyperpolarized potentials by benzoylaconine in guines-pig cardiac myocytes.
Material and methods
Cell isolation
Single ventricular cells were isolated from guinea pig heart according to Reference (3). Briefly, a guinea pig weighing 250 to 350 g was anesthetized with pentobarbital sodium (approx. 30 mg/kg,, IP); the heart was removed and cannulated through the aorta on a Langendorff apparatus warmed at
37°C. The heart was perfused first with Tyrode's solution at 37°C for 3 minutes, second with nominallyCa2+-free Tyrode's solution for 5-7 minutes, and finally with collagenase (0.12-0.20 mg/ml) in nominallyCa2+-free Tyrode's solution, which is recirculated using a rotor pump for 10-15 minutes. Most of the myocytes dissociated with collagenase were rod-shaped and quiescent in the storage solution , and were Ca2+-tolerant in the Tyrode solution. Tyrode's solution contained (mmol/L) NaCl 143; KCl 5.4; CaCl2 1.8; MgCl2 0.5; NaH2PO4 0.25; HEPES 5; pH to 7.4 with NaOH. Storage solution: KOH 70, L-glutamic acid 50, KCl 40, taurine 20, KH2PO4 20, MgCl2 3, glucose 1, HEPES 10, EGTA 0.5, pH to 7.4 with KOH. The percentage of rod-shaped cells of Ca2+-tolerant were 70.4+2.6 in the Tyrode solution.
Solution and membrane current measurements
Membrane currents were recorded using the patch-clamp technique in the whole-cell configuration with a AXON 200B(USA)patch-clamp amplifier. Ventricular myocytes were voltage-clamped with fire-polished patch pipettes (tip resistance, 1 + 3 M) filled with a solution containing (mmol/L) CsF 145, NaF 5, HEPES 5, pH 7.2 adjusted with CsOH. The bath solution contained the following composition (in mM): NaCl 5-15, MgCl2 1.2, CsCl 5, CaCl2 1.8, teramethylammonium chloride (TMA-Cl) 125 glucose 11 HEPES 20, pH 7.4 adjustedwith TMA-OH. The choice of solutions included internal F- which blocks Ca2+ current and internal and external Cs+ which blocks inward rectifier K+ current thus leaving only TTX-sensitive INa(4).
As a routine protocol for INa recordigns, Holing potential was -140 mV and test voltages ranged from -80 to +30 mV in +10 mV increments and were delivered at a frequency of 1 Hz in all experiments. The time constant of the capacitative transient was 296+14 ^.S and the membrane capacitance was calculated to be 101+6 pF Mean series resistance (Rs) was 5+1 M. All experiments were conducted at room temperature of 22 °C to 24°C.
Data acquisition and analysis.
Data were filtered at 3 kHz, digitized at 10 KHz by using PCLAMP software and Digidata 1200 A/B AD/DA card (Axon Instruments,Inc.) and stored on a IBM At personal computer. Original current recordings were leak and capacity corrected either during an experiment wsing a P+P/n protocol or off-line using a scaled current recording in which no membrane current was evident. The values for Na+ conductance (GNa) were calculated according to the equation: GNa=I Na /(V t -Vrev), where INa is asolute value of Na+ current, V t is test potential and Vrev is reversal potential.. The values of GNa and V t -Vrev were then fitted to a Boltzmann distrution from which midpoint (Vm) and slope factor (s) were calculated according to the equation: GNa/G max =1/{1+exp[(Vm - V)/s]}, where V is a test potential and Gmax is the maximal conductance value of each current-voltage relation extrapolated through the pseudo-reversal potential (Vrev) estimated by linear regression of currents at the most positive potentials. The fitted curves for steady state INa availability (ha>) were obtained using the following form of the Boltzmann equation: ha> =I/Imax= 1/{1+exp[(V - V1/2)/k]}, where V1/2 is the voltage at half inactivation, k is the slope factor, V is test voltage.
Statistics Results are given as mean+SD. A paired t test was used for significance, and a value of p < 0.05 was considered statistically significant.
Results
Effects of benzoylaconine on Na current
Comparing the effect of benzoylaconine (100 uM*L_1) on cardiac INa, under control condition, INa was elicited by depolarizations to -55 mV or to -60 mV from holding potential of -140 mV, the maximal inward peak at -40 mV(60.33±4.69,pA/pF) and its apparent reversal potential (5 mV). Current activared and inactivated rapidly and completely, leaving no steady-state component at the end of a 50 mS depolarization.
After 5 min of exposure to benzoylaconine (100 uM*L_1), peak INa amplitudewas increased, the maximal peak current was increased and reversal potential not changed. INa was elicited by depolarizations to -60 mV or to -70 mV from holding potential of -140 mV. The current failed to inactivate fully at all step-voltages. The peak current-voltage (I/V) relationships obtained from the
above experiment is shown in panel B under control conditions (squares) and during exposure to benzoylaconine. Results were similar to those obtained in 11 other cells.
Effect of benzoylaconine on the voltage dependence of GNa
The threshold for activation to benzoylaconine was shifted to left about -70 mV and maximal Gmax increased by (21% and 26%). After the normalized conductance were fitted according to the boltzmann distribution, benzoylaconine induced an 7 mV shift of the conductance corve toward hyporpolarized potentials at the curve of GNa/voltage-relationship and did not significantly change the slope factors (8.33 to 8.38 8.45 mV). The conductance half-point value (Vmid) was shifted in the hyperpolarizing direction from -40 mV for control data to -47 and -49 mV after addition of benzoylaconine.
Effect of benzoylaconineon characteristics of slow inactivation
To examine the effect of the voltage dependence of the inactivation, we usedto the voltage protocol of two-pulse to obtained the data. Benzoylaconine increased the magnitude of INa at potentials where it was maxinal (-140 to -100 mV). In addition, there was a small steady-state or non-inactivating component present at potentials where it was -40 to -20 mV. After the data from the experiment was fitted according to the benzoylaconine caused a negative shift of the half-availability value (V1/2 ) at which half of the Na+ channels were inactivated from -84.02 mV to -91.71 -93.55mV. The slope factor (k) were significantly increased from 4.59 to 5.71 5.82 mV and leaves about 10% a steady-state, non-inactivating levels of the slow current componentat at low test potentials. Similar results were observed in eight patches.
Discussion
It is also reported voltage-gated Na channels would markedly affect cardiac excitability in normal heart muscle. These effects include either Na+ influx and subsequent Na+ loading via the slowly inactivating INa enhanced the Na-Ca exchange to cause an increase in contractive force or prolongation of action potential result in a positive inotropic effect by increasing Ca2+ influx via the Ca2+ current as well as via the electrogenic Na-Ca exchanger(4)' We have reported that several effect of benzoylaconineon myocadial cells(1'2). Among them , it was described that the benzoylaconineprolonged the duration of action potential, increased the maximum rise of the action potential upstroke (Vmax) and suspected to be a Na+ channel modefier. The susceptibility of cardiac Na+ channels was analyzed in order to obtain a closer insight into their gating and kinetic properties.
Our results shown that the most striking effects of benzoylaconine(100 uM*L_1) cardiac W were the slowing of INa decay and the increase in peak currentD All of first, The simplest explanation for the two effects is that some of the channels that open during the given depolarization maintain a finite probability of being open and would lead to a progressive increase in the total number of open Na+ channels during a prolonged depolarization, including the time at which maximal current is flowing. On the other hand, the fact that Gmax increased by about 21% which must be the result of a greater number of channels open at each test voltage, In addtion, the increase in maximal INa would be attributable to the shift in conductande which the consequent increase in the electrochemical driving force is sufficient to account for the maximal 21% increase in peak current with 10 - 15 mmol/L extracellular Na+ and Erev of 5 mV.
Other strong effects are the inactivation process of cardiac INa. It was apparent that The inactivation time courses were considerably slowed and steady state currents were present during a prolonged depolarization. benzoylaconine caused about 9 % channels to fails to be inactivated at given conditioning potentials that were maintained for 1000 mS. In addition, the slope factor was increased and V1/2 was shifted to more negative potentials, which seems additionally to interfere with the voltage control of channel gating and appeared to result from the opening of previously inactivated Na channels. There results imply that the voltage dependent of inactivative process in benzoylaconine-modified Na channels has undergone some important alterationsD These effects strongly resemble in which reported that BTX and APA modified Na channels of cardiac(4'5).
According to the above results, it may be convenient to explain the mechanism according to the model reported(7). In this scheme, interconversions between the O (open), Ii (first inactivated I)
and I2 (second inactivated ) states take place in normal Na channels according to voltage-dependent kinetic rates and I2 (second inactivation process) is essentially irreversible to I1 (first inactivated state) rapidly. Most channels will be in I2 after long pules. The process of slow inactivation by the toxin-induced can be explained that incresing the free energies of the first and second inactivared states, as well as the transition energies for the O--I1. These altered energetics will selectively populate O at the expense of the inactivated channels, which produce a increase in burst duration of toxin-modified channels, whose formation is also slowed. In adddition, Recovery of inactivation seems that the transitions of resting state (R) is stronly modified, after repolarization, a process that normally has a delay as I2 converts to Ii become no delay after toxin-modified. It is suggested that because less of he I2 state formed and this is converted to I1 relatively rapidly.
Removal of Na inactivation may be an important antiarrhythmic priciple in geart muscle and represents an new mode of actoin of drugs chassified by Vaughan Williamsas class 3 antiarrhythmics. The class is rather formally defined since prolongation of the action potential as the typical feature is ambiguous in nature and can result from both an increase of inward currents or a decrease of outward currents. Benzoylaconine-modified Na channels must be expected to be exclusively responsible for the prolongation of the action potential. Thus, Na channel modification reduces cardiac excitability during diastole by prolonging the absolute refractory period. We believe that future experiments using single channel analysis may resolve a simple dinetic model for modified Na channels. Reference
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