| Objective: Patch clamp technique, built by Neher and Sackmann in 1976 was used for study the action of ionic channels molecule of cellular membrane by recording the current through ionic channel, which is the basic excitation unit of neurocyte cellular membrane, tunica muscularis, glandular organ and many other tissues and basic of bioelectricity activity. The ionic channels possess considerable physiologic function, for example, they can create and conduct the electrical signal. Especially, active potential creating, exciting and contraction linkage of myocardial cells are baced on the kinds of ionic channels open or close. Most of antiarrhythmic drugs produce a marked effect on the ionic channels. Studies in animal models have shown that theα-adreneergic acceptor andβ- adreneergic acceptor systems play a synergistic role in the genesis of arrhythmias. However, during ischemia and reperfusion,α-adreneergic stimulation may be particularly arrhythmogenic. therefore, we suppose that phentolamine as aα-adreneergic acceptor blocking pharmacon maybe play the opposite role of the antiarrhythmic effect. This experiment objective is recording the rat ventricular myocyte current of the sodium Channels between the control group and the phentolamine group using the patch-clamp technique, then we discuss if the phentolamine has the antiarrhythmic effect.Material and methods:1 Experimental animals: SD rat of either sex weighting 150～200g were provided by the Experimental Animal Center of Hebei Medical University, China.2 Cell separation: Single rat ventricular myocytes were isolated enzymatically(collagenase, Gibco Corporation),normal ventricular myocytes are the control goup before using the phentolamine ,and after using the phentolamine in the extracellular fluid are the experiment group.3 Current record: Transmembrane currents (INa )were recorded with the whole-cell patch clamp technique, using EPC-9 patch clamp amplifier. Data acquisition and processing were performed by the pulse-pulsefit software.Then,the currents were compared between the experiment group(phentolamine group)and the control groups.4 Statistics:Data were expressed as mean value±standard deviation ( x±s). The statistical analysis was performed using SPSS,versin 13.0. The variance within groups was analyzed by One-Way ANOVA (SPSS,versin 13.0), A value of P < 0.05 was considered statistically significant.Result1 Peak INa current density(pA/pF) between the phentolamine group and the control groupPhentolamine decreased INa current density without changing the threshold, peak and reversal potentials. The peak INa current density was down-regulated in ventricular myocytes from phentolamine group compared with control group, This was not accompanied by a shift in the INa current density-voltage relation . INa were elicited in response to depolarizing pulses positive to -70 mV, reaching a maximal value around -40 mV and reversing at about +50 mV.At test potential of -40 mV, the peak INa current density(pA/pF) among the control group , the phentolamine(10-5) group and the phentolamine(10-6) group were -46.92±2.16pA/pF (n=10) , -16.64±0.84pA/pF (n=10) and -22.62±0.84 pA/pF (n=10), respectively. The differences between the two phentolamine groups and the control group were both significant (P<0.05).The difference between phentolamine(10-5) group and the phentolamine(10-6) was also significant (P<0.05).2 INa current steady-inactivation curves between the phentolamine group and the control groupThe Half-inactivation potential(V0.5) of the availability curve(I/Imax curve)was shifted significantly in the hyperpolarizing direction in the two phentolamine(10-5,10-6) groups compared with control group (-74.24±0.43mV,n=10), P<0.05, The Half-inactivation potential(V0.5) of the phentolamine(10-5) and phentolamine(10-6) were -105.33±1.09 mV(n=10),-95.32±1.10 mV(n=10)respectively,and the difference between the two experiment groups was significant (P<0.05).3 INa recovery curves from inactivation between the phentolamine group and the control groupThe time-constant (τ) of INa recovery curves from inactivation among the three groups (control, phentolamine10-5 and phentolamine10-6) were 70.62±0.61ms (n=16), 124.3±1.27ms (n=12), 104.58±1.14ms (n=12)respectively . the two hentolamine groups vs control group, P<0.05,and the difference between the phentolamine10-5 and phentolamine10-6 was significant (P<0.05).Conclusions1 The animal experiment indicated that phentolamine induces significant down-regulation peak of INa current density,reduces the time of INa inactivation, delays the time of INa recovery from inactivation. These phenomenon indicate that phentolamine can decrease in the velocity and amplitude of phase 0 of action potential,abnormal transmembrane action potentials,degrade the ventricular myocyte excitability and contribute to inhabit ventricular arrhythmias.2 The peak INa current density was down-regulated ,half-inactivation potential(V0.5) of the INa was up-regulated ,the time-constant (τ) of INa recovery curves from inactivation was long-regulated in ventricular myocytes from phentolamine group compared with control group. These differences between the control group and the phentolamine group is significan(P<0.05).3 Abroad and domestic studies have shown thatα-adreneergic stimulation may be particularly arrhythmogenic during ischemia and reperfusion. Phentolamine as aα-adreneergic acceptor blocking pharmacon maybe play the opposite role of the antiarrhythmic effect, however it dose not have evidences that phentolamine effect on the ionic channel or have the antiarrhythmic effect. This study indicate that phentolamine inhibits the fast INa current in phase 0 of action potential.
|
PDF Full Text Request