Determination The Contents Of Bufalin In Chan Su,and Detecting Its Effects On Calcium Signals In Rat Ventricular Myocytes

Chan Su,a white secretion prepared from both the auricular and skin glands of Chinese toads,is a traditional Chinese medicine and has been widely used in East Asian countries.Chan Su has been proved to have strong pharmacological activities,such as the activity of antitumor,diuretic,analgesic,and anti-inflammatory.In recent years,the compounds of Chan Su have been proved to have significant curative effect in the treatment of cardiovascular diseases.Bufalin is a bufanolide steroid compound in Chan Su.From our study,we examined bufalin is exactly one of the main components of Chan Su by the High performance liquid chromatography?HPLC?.Therefore,we assumed bufalin may have the pharmacological activity of cardioprotection.In our research,the content of bufalin in Chan Su was tested by HPLC.We examined the impacts of bufalin on I_Ca-La-L by Whole cell Patch Clamp technique,and we detected the cardiocyte contractility by IonOptix system.Objectives:To examine the content of bufalin in Chan Su,the influences of bufalin on L-type calcium currents and contractility in rat ventricular myocytes.Methods:In this study,we determined the content of bufalin in Chan Su by HPLC.We explored the influence of bufalin on L-type calcium currents by Whole cell Patch Clamp technique.We detected the contractility in rat ventricular myocytes by IonOptix system.Results:1 The contents of bufalin in Chan SuWe used High performance liquid chromatography method?HPLC?with a reversed phase Altima column?250mm x 4.6mm,5mm?,0.5%potassium dihydrogen phosphate acetonitrile?50:50??Adjusted pH:3.25±0.02 by phosphoric acid?as mobile phase,detection wavelength:296nm,column temperature:40?,flow rate:1.0ml/min.According to the standard curve calculation,we found the content of bufalin in Chan Su is about 3%.2 Confirmation of I_Ca-Lin rat ventricular myocytesThe currents were almost abolished by nicardipine?0.01 mM?,which indicated that these currents were Ca²⁺currents.?P<0.01?.As a specific T-type Ca²⁺channel blocker,NiCl₂?0.1 mM?did not affect the currents,indicating that there is no contamination of T-type Ca²⁺currents.Application of verapamil?10?M?,a specific L-type Ca²⁺channel blocker,could almost completely abolish the currents?P<0.01?,which indicated that these currents were L-type Ca²⁺currents.3 Effects of bufalin on I_Ca-LAfter exposure to 300?M bufalin,the peak of I_Ca-La-L was apparently depressed?P<0.01?.The I_Ca-La-L remained stable after washing with external solution.Figure 3a,b shows that bufalin can inhibit the I_Ca-L,and this inhibition is irreversible.Figure 3c is a time course of 300?M and washout.Representative current recordings with the activation protocol at the sequential administration of bufalin?1-300?M?are shown in Figure3d,e.Bufalin progressively suppressed I_Ca-La-L in a dose-dependent fashion.The equation indicates that the half-maximal inhibitory concentration(IC₅₀)of bufalin is 60?M?Figure 3f?.Figure 3g and H shows that bufalin?300?M?can also inhibit the I_Ca-La-L which were excited by calcium agonist Bay K8644?10?M?.4 Effects of bufalin on I-V relationship of I_Ca-LThe effects of bufalin?3-300?M?on the I-V relationship of the I_Ca-La-L is shown in Figure 4.Figure 4a shows representative trace recordings with the sequential treatments of?3-300?M?.Figure 4b is the currentdensity-voltage relationship with controls for?3-300?M?bufalin and verapamil?10?M?.These results indicated that?3-300?M?bufalin could significantly depress the maximum current.5 Effects of bufalin on steady state activation and inactivation of I_Ca-LThe voltage dependence of steady state activation and inactivation of I_Ca-La-L in the presence or absence of bufalin is shown in Figure 5.Values at V_1/2,and the slope factor?k?of the normalized activation conductance curves for control,3 and 30?M were 3.52±1.13 mV/8.78±0.76,0.73±1.29 mV/9.17±0.95 and 2.34±1.41 mV/9.38±0.97?Figure 5A?.The V_1/2/2 and the k value of the steady state inactivation curves for control,3 and 30?M was-24.60±0.16mV/4.79±0.13,-27.38±0.10 mV/5.59±0.09,-31.10±0.16 mV/5.97±0.15respectively?Figure 5b?.The intergroup differences were compared by the half-inactive voltage(V_1/2).The 3?M bufalin did not shift the steady state inactivation of I_Ca-L?P>0.05?,and the 30?M bufalin could shift the steady state inactivation of I_Ca-L?P<0.05?,vs.control.6 Effects of bufalin on myocyte shorteningRepresentative data of cell shortening were recorded before and after administration of bufalin?100?M;Figure 6?.These results showed that bufalin could restrain cell shortening by 33.83±4.01%at 100?M?P<0.01?.Conclusions:Our study examined the content of bufalin in Chan Su and explored the electrophysiological mechanisms of bufalin on its cardiopr-otective effects.The results showed that the content of bufalin in Chan Su is about 3%,bufalin had significant and dose dependent inhibitory effects on I_Ca-L.It reduces cell contraction in ventricular myocytes.These results may provide a theoretical basis for bufalin on its further clinical practice.