The Effects Of Emodin On L-type Calcium Channel Of Guinea-pig Smooth Muscle

Objective: The research of gastrointestinal dynamics and its related disease is anemerging science and develops rapidly in recent years. To find out the etiology ofgastrointestinal motility disorders is of great significance to treat the diseases of thedigestive tract motility disorders. The study to reveal the influence of Chinese medicineprescription Da Cheng Qi Tang on gastric motility and the mechanisms of it regulatinggastric motility was less. The aim of this paper was to study the effect of its main activeingredient Emodin on L-type calcium channelcurrent （ICa） which located on smoothmuscle cell membrane of gastric antral in guinea pig, and to explore the mechanism of iton regulation of the gastrointestinal motility.Methods: The traditional whole-cell patch clamp technique was introduced torecord the L-type calcium current （ICa） of normal gastric antral smooth muscle cells ofguinea pigs. After using Ba2+as charge carrier, Ba2+current (I_Ba) can be recorded.Respectively using the superfusing cells containing the solution ofBa2+-PSS,10μMEmodin PSS and that of20μM Emodin PSS, The effect of Emodin onI_Bawas observed on gastric antrum amooth muscle cells of guinea pigs. I_Bacurrent peakand the I_BaI-v curve as well as I_Basteady activation and inactivation characteristics.Weneeded more than six sets of complete data in each group.Results: Under the traditional whole-cell patch clamp record mode,Continueously perfusing cells with the extracellular fluid containing Ba2+, clamp downmembrane potential on-80mV, give single polarization pulse stimulation with the timecourse of400ms and phase step directly from-80mV to0mV, and this stimulationrepeated every10s. In the experiments we recorded several single ICacurrent. When thecurrent stay stable, Add different concerntation Emodin to the extracellular fluid. I_Bacould be significantly activated which was time-dependent, and I_Bacurrent peak wasobviously increased in both of the two groups with10μMEmodin and20μMEmodin compared with the control group that did not contain emodin.The experimental results showed that the influence of Emodin with differentconcentrations on I_Bawas voltage-dependent. If10μmol Emodin was added to theextracellular fluid, I_Bacurrent could be apparently activated in-10mV⁺30mV voltagerange（*P<0.05,**P<0.01,n=10）, while if20μmol Emodin was added to the extracellularfluid, I_Bacurrent could be apparently activated in-20mV⁺40mV voltage range（*P<0.05,**P <0.01, n=10）. At the same time,10μM Emodin and20μMEmodin have nosignificant influence on the activation threshold value of I_Bachannel.Clamp membrane potential on-80mV, adopt the polarization pulse stimulationwith the time course of400ms, from-60mV to+60mV, steps order potential as10mV,aseries of current value correspond to different membrane potential were received. Makea curve fitting on current value ratio correspond to different membrane potential withBoltzmann equation I/Imax=1/{1+exp [（VmVa1/2）/κ]}, and get the steady-state activatecurve before and after medicine. Va1/2was half of the largest activated voltage, κ wasslope factor. After joining10μMEmodin Emodin, Va1/2changed from-12.91±0.61mVinto-12.69±0.50mV （n=10, P>0.05）, slope factor κ was6.61±0.53and5.47±0.44（n=10,P>0.05）, it showedthat10μM Emodin had no significant influence onthe steady-state activation of I_Bachannel.Clamp membrane potential on-80mV.First give the depolarized condition pulsestimulation with the time course of4s, and phase step from-100mV to+40mV withevery step order potential as20mV. After conditions pulse stimulation, I_Bachannelcould be completely in the deactivation state. After conditions pulse stimulation andgiving the time course of1s with fixed test pulse stimulation depolarizing to0mV, aseries of I_Bachannel inactivation current could be received. Use Boltzmann equation I/IMax=1/{1+exp [（Vm-Vi1/2）/k]} to fit the ratio of the current and the maximumcurrent （I/Imax） under different testing potential. Vi1/2was half the deactivationvoltage, κ was slope factor. Do statistical analysis of Vi1/2, the results suggested thatafter adding Emodin, Vi1/2changed from-27.86±0.76mV into-37.23±0.82mV（n=10,P <0.05）, slope factor κ was10.64±0.65and11.73±0.75（n=10,P>0.05）.Itshowed that10μM Emodin could make the steady inactivation curve of I_Bamove to thethe negative direction of voltage, that is,10μMEmodin accelerated the inactivationprocess of I_Bachannel.Conclusion: Both of the10μMEmodin and20μMEmodin have remarkableactivation effect on L-type calcium current （ICa） with voltage-dependence andconcentration dependence.10μMEmodin have no significant influence.on the steady-state activation of I_Bachannel, but can make the steady inactivation curve of I_Bamove to the the negative direction of voltage.