| Objective:To investigate the effects of ligustrazine(TMP)and tanshinone IIA on currents and kinetic characteristics of sodium ion channel in ventricular myocytes of SD rats,and to discuss the changes of channel currents and kinetic processes after combined administration of the two drugs.Methods:Firstly,SD rat heart was cut rapidly after anesthesia at room temperature(about 25 ?)put in an ice bath quickly.Secondly,single ventricular myocytes were obtained by reverse perfusion of Langendorff aorta.Then,the sodium ion current was recorded by whole-cell patch clamp technique.The drugs were added accumulated in the way of extracellular fluid perfusion,the changes of sodium current after different doses of TMP and TanshinoneIIA were recorded,then the changes of kinetic characteristics of sodium channel current were analyzed.Results:1.Effects of TMP on INa.(1)Concentration dependence of TMPINa was inhibited by TMP in a concentration-dependent manner.When the concentration of TMP was lower than 20 ?M,it has no significant effect on INa.When it was over 50 ?M,with the increase of drug concentration,the inhibition of current was also enhanced.Under the influence of 50 ?M and 200 ?M TMP,INa-Peak decreased from(-81.32±3.28)pA/pF to(-58.10±4.16)pA/pF and(-35.65±3.23)pA/pF(P<0.01,n=6).(2)Effects of TMP on the Current-Voltage relationship(I-V)curve of INaUnder the influence of TMP which was in the range of 50?400 ?M,the I-V curve of INa moved up significantly.However,the track trend of I-V curve have not been changed.(3)Effects of TMP on activation kinetics of INaIn the range of 50?400 pM,the steady-state activation curve shifted to the right under the influence of TMP.the V1/2-ac was changed from(-42.92±0.24)mV to(-41.20±0.19)mV and(-37.6±0.17)mV(P>0.05,n=6)under the influence of 50 ?M and 200 ?M TMP.That is to say,the activation of the channels wasn't been affected by TMP.(4)Effects of TMP on inactivation kinetics of INaThe steady-state inactivation curve of INa shifted to the left under the influence of TMP in the range of 50?400 ?M,50 ?M and 200 ?M TMP changed the V1/2-in from(-57.71±0.21)mV to(-64.05±0.41)mV and(-69.85±0.3 9)mV respectively(P>0 05,n=6).That is,TMP has no significant effect on channel inactivation process.(5)Effects of TMP on recovery kinetics of INaThe recovery curve of INa shifted to the left under the influence of TMP in the range of 50?400?M,50 ?M and 200 ?M TMP changed ? from(20.88±1.59)ms to(18.65±1.38)ms and(15.62±0.8)ms(P>0.05,n=6).That is to say,TMP has no significant effect on channel recovery process.2.Effects of Tanshinone IIA on INa.(1)Concentration dependence of Tanshinone IIAINa was inhibited by Tanshinone IIA in a concentration-dependent manner.There was no significant effect on INa lower than 10?M of Tanshinone IIA.When it was over 20 ?M,with the increase of drug concentration,the inhibition of current was also enhanced.Under the influence of 20 ?M and 80 ?M Tanshinone IIA,INa-Peak decreased from(-110.61±6.28)pA/pF to(-87.?8±5.76)pA/pFand(-54.53±3.15)pA/pF(P<0.01,n=6).(2)Effects of Tanshinone IIA on the Current-Voltage relationship(I-V)curve of INaUnder the influence of Tanshinone IIA in the range of 20?160 ?M,the I-V curve of INa moved up significantly.However,the track trend of I-V curve have not been changed.(3)Effects of Tanshinone IIA on activation kinetics of INaUnder the influence of Tanshinone IIA in the range of 20?160 ?M,there was no significant effect on the steady-state activation curve of INa,V1/2-ac was changed from(-43.85±8.07)mV to(-43.95±2.44)mV and(-43.99±0.79)mV(P>0.05,n=6)when the concentration of Tanshinone IIA were 20 ?M and 80 ?M.(4)Effects of Tanshinone IIA on inactivation kinetics of INaThe steady-state inactivation curve of INa shifted to the left significantly under the influence of Tanshinone IIA in the range of 20?160 ?M,20 ?M and 80 ?M Tanshinone IIA changed theV1/2-in from(-52.15±0.96)mV to(-64.87±0.99)mV and(-75.71=0.75)mV(P<0.05,n=6).That is,Tanshinone IIA madeV1/2-in move towards hyperpolarization,which accelerated channel inactivation.(5)Effects of Tanshinone IIA on recovery kinetics of INaThe recovery curve of INa shifted to the right under the influence of Tanshinone IIA in the range of 20?160 ?M,20 ?M and 80 ?M Tanshinone IIA changed ? from(11.52±1.18)ms to(20.84±0.99)ms and(30.81±1.01)ms(P<0.05,n=6).That is,Tanshinone IIA can delay the recovery process of channels.3.Effects of combined applications of Tanshinone IIA and TMP on INa(1)Concentration dependence of Tanshinone IIAINa was inhibited by Tanshinone IIA+TMP in a concentration-dependent manner.In order to preliminarily study the effects of these two drugs combination and facilitate lateral control,four concentrations combinations of Tanshinone IIA+TMP were set up,(20+50),(40+100),(80+200)and(160+400)?M.The results showed that the inhibitory effect of the combined drug on INa was enhanced with the increase of the concentration gradient.When the concentration was(20+50)?M and(80+200)?M,INa-Peak decreased from(-103.94±6.58)pA/pF to(-59.86±5.12)pA/pF and(-30.60±4.23)pA/pF(P<0.01,n=6).Which showed an obvious concentration-dependent inhibition.Compared with the two situations above,the inhibition rate of INa in combination was higher.For example,the inhibition rates of 50?M TMP,20?MTanshinoneIIA and(20+50)?M TanshinonelIA+TMP were(29.1±1.1)%,(19.1±1.1)%and(42.4±1.4)%(P<0.05,n=6)respectively,which highlighted the advantages of the two drugs combined use.(2)Effects of combined applications of Tanshinone IIA and TMP on the Current-Voltage relationship(I-V)curve of INaAs in the case of the above alone administration,Under the influence of combined drugs,the I-V curve of INa moved up significantly.(20+50)?M and(80+200)?M Tanshinone IIA+TMP changed VPeak from-30 mV to-25 mV and-20 mV(P<0.01,n= 6).However,the track trend of I-V curve have not been changed.(3)Effects of combined applications of Tanshinone IIA and TMP on activation kinetics of INaThe steady-state activation curve significantly shifted to the right,the(20+50)?M and(80+200)?M combined drugs made V1/2-ac changed from(-42.74±0.43)mV to(-42.32±0.4)mV and(-39.9±0.75)mV(P>0.05,n=6).That is,the activation of the channels wasn't been affected by combined drugs.(4)Effects of combined applications of Tanshinone IIA and TMP on inactivation kinetics of INaThe steady-state inactivation curve of INa shifted to the left significantly under the influence of combined drugs,(20+50)?M and(80+200)?M drugs changed theV1/2-in from(-81.61±0.72)mV to(-82.89±0.59)mV and(-88.21±0.49)mV(P>0.05,n=6).That is,the combined drugs had no significant effect on channel inactivation process.By comparing the changes of V1/2-in in the above three cases,it was found that the combination of the two drugs had little effect on channel inactivation process.(5)Effects of combined applications of Tanshinone IIA and TMP on recovery kinetics of INaThe recovery curve of INa shifted to the right under the influence of combined drugs,(20+50)?M and(80+200)?M drugs changed ? from(20.47±0.84)ms to(30.22±0.97)ms and(31.62±1.02)ms(P<0.05,n=6).That is,Tanshinone IIA+TMP can delay the recovery process of channels.By comparing the changes of ? in the three cases,it was found that the combined group could reverse the acceleration of channel recovery by TMP,and had less effect on the recovery process of the channel compared with using Tanshinone IIA only.Conclusion:TMP,Tanshinone IIA and combined use of two drugs inhibited INa in SD rat ventricular myocytes in a dose-dependent manner respectively,and the inhibition effect was better in combined use group.Moreover,combined use of two drugs had minor effect on the kinetic characteristics of the channel. |
PDF Full Text Request