Inhibitory Effects Of Daurisoline On Cav1.2Calcium Channel Of Murine Ventricular CMs

Aim:Daurinsoline (DS) is extracted from the root of Menispermum dauricum DC, the bisbenzyl isoquinoline alkaloids. In this study, we investigated the effects of DS in transfected HEK293cells with Cav1.2and Cav3.1calcium channel expression and Cav1.2calcium channel of ventricular cardic myocytes (CMs) during murine cardiac embryogenesis.Methods:1. The whole-cell patch clamp technique were used to record ICav1.2and ICav3.1expressed in transfected HEK293cells and investigate the effects of DS on electrophysiological characteristics of Cav1.2and Cav3.1.2. Adult and embryonic ventricular CMs of murine were isolated by enzymic method to investigate the effects of DS on ICav1.2during murine cardiac embryogenesis by using whole-cell patch clamp technique.Results:1. DS at1,3and10μmol·L-1could inhibit ICav1.2expressed in transfected HEK293cells in a dose-dependent manner. The inhibition rates of DS at3Lmol·L-1on ICav1.2was32.37±6.63%and significant different compared with control (p<0.001, n=6). When compared with isradipine (isradipine, ISR)(3μumol·L-1), the blocker of Cav1.2(83.38±2.27%.), were significant different (p<0.001, n=6).2. DS at1,3and10μmol·L-1could inhibit ICav3.1expressed in transfected HEK293cells in a dose-dependent and voltage-independent manner. At different holding potentiols (HPs)(-110mV and-70mV), the inhibition rates of DS at3μmol·L-1on ICav3.1were37.92±5.76%and45.29±8.72%, respectively. There were no significant differences at different HPs (p>0.05, n=6). At HPs-110mV and-70mV, the inhibition rates of mibefradil3μmol·L-1), the blocker of Cav3.1, were50.52±7.23%and94.80±1.62%, respectively.The inhibitory effects of mibefradil holding at-110mV and-70mV were significant differences (p<0.001, n=6). There were no significant differences between DS (3μmol·L-1) and midefradil (3μmol·L-1) at HP-110mV (p>0.05, n=6), but less potent at HP-70mV(p<0.001,n=6).3. At the same HP (-110mV or-70mV) DS did not significantly change the value of τinact before and after administration in transfected HEK293cells expressed T-type calclium channel Cav3.1(p>0.05, n=6).4. DS at1,3and10μmol·L-1could inhibit ICav1.2on adult ventricular CMs in a dose-dependent manner. The inhibition rates of DS at3μmol·L-1on ICav1.2was50.17■4.51%and significant different compared with control (p<0.001, n=6). When compared with isradipine (3μmol·L-1), the blocker of Cav1.2(81.75±2.02%), were also significant different (p<0.001, n=6). DS3μmol·L-1could shift the inactivation curve left and the recover curve to the right.5. DS3μmol·L-1could inhibit ICav1.2on embryonic ventricular CMs and the inhibition rates of DS at3μmol·L-1on ICav1.2was52.38±2.44%and significant different compared with control (p<0.001, n=5). When campared with isradipine?(3μmol·L-1), the blocker of Cav1.2(73.94±3.24%), were significant different (p<0.001,n=5). Conclusion:1. DS could inhibit the ICav1.2and ICav3.1expressed in transfected HEK293cells in a dose-dependent manner and inhibitory effects of DS were less potent than calcium channel blockers at the same concentration,p<0.001.2. The inhibition of mibefradil on ICav3.1was determined by HP, the more depolarized HP, the more potent. However, the inhibitory effects of DS had no relationship with HP.3. At the same HP (-110mV or-70mV) DS did not influence the inactivation of T-type Cav3.1calcium channel in transfected HEK293cells before and after administration, p>0.05.4. DS could inhibit ICav1.2of adult ventricular CMs in a dose-dependent manner and less potent campared with Cav1.2blocker, isradipine,p<0.001. DS could also accelerate the rate of inactivation and delay the recovery time from inactivation of Cav1.2.5. DS could also significantly inhibit ICa1.2on embryonic ventricular CMs (15.5-18.5d) and less potent campared with Cav1.2blocker, isradipine,p<0.001, which indicated that DS could inhibite ICav1.2during murine cardiac embryogenesis.