Effects Of U-73122 And Tetrandrine On Store-operated Ca²⁺ Channels Of Hepatocytes After Hepatic Ischemia-reperfusion Injury In Rats

Objectives:This experiment is to improve and fine-tune the method in isolating Sprague-Dawley(SD)rat hepatocytes which settles for patch-clamp recording techniques and in establishing the hepatic ischemia reperfusion injure(HIRI)model. It's using patch-clamp techniques to study the characteristics of store-operated Ca²⁺ channel currents(Isoc)in freshly isolated hepatocytes of SD rat in physiological and HIRI conditions;to research the change of Isoc after HIRI and the effects of U-73122 and tetrandrine(Tet)to that process.Methods:hepatocytes were obtained from SD rat with in situ typeâ…£collagenase perfusion.We added 10mM EGTA and 2μM thapsigargin in intracellular solution to actives SOC and study Isoc of hepatocytes by whole-cell patch-clamp technique. Using the method of clamping the portal vein and hepatic artery of the median lobe and left lateral lobe for 30 minutes and reperfusion for 40 minutes,we established the model of hepatic ischemia/reperfusion injury which settles for patch-clamp recording techniques,also we isolated the hepatocytes for patch-clamp experiments.U-73122 (at 0.3μM,1μM,3μM,10μM and 30μM)and Tet(at 3μM,10μM,30μM,100μM and 150μM)were added to the normal and ischemia/reperfusion hepatocytes with rapidly solution changer to investigate the effects of U-73122 and Tet on I_SOC.Results:The improved method in isolating hepatocytes of SD rat was much easier to operate and required less collagenases.The hepatocytes obtained were 3.8×10⁷/g with the purity of 91%and viability of 89.6%on average,and these cells were suitable for subsequent patch clamp experiments.To actives SOC we added 10 mM EGTA and 2μM thapsigargin in intracellular solution.We could record obvious I_SOC and calcium-dependent Cl^- current,which was stable in 20 min without rundown.The cell was clamped at 0 mV,then applied a series 200 ms pulses from -100 mV to +80 mV in 20 mV increments at a frequency of 0.5 Hz.The results demonstrated that the peak amplitude of I_SOCwas -195.84±17.19pA(n=15)at -100 mV in normal hepatocytes;theâ… -â…¤curve of I_SOCreflected inwardly rectifying at negative potentials and calcium-dependent Cl^- currents performed out rectifying at positive potentials with reversal potential closing to 0 mV.Clamping the portal vein and hepatic artery of the median lobe and left lobe for 30 minutes with subsequent reperfusing for 40 minutes can successfully establish the model of HIRI with high survival percentage of rat.The hepatocytes obtained in the model had distinct morphological differences with normal cells,and the hepatocytes obtained were 3.0×10⁷/g with the purity of 85.3%and viability of 78.2%on average.Those hepatocytes were some easy to attach,form giga seal and rupture in the subsequent patch clamp research.Using the same protocol,the results illustrated that clamped at -100 mV the peak amplitude of I_SOCwas -447.31±26.04pA(n=15)in HIRI hepatocytes,and the peak ameliorate of I_SOCwas -201.91±23.41pA(n=15)in control cells.As a result,there was prominent statistical discrepancy between them(n=15,P＜0.01),but the characteristics ofâ… -â…¤curve and the reversal potential did not change compared with control.After detecting the effects of different drugs on HIRI hepatocytes,we found that U-73122 (at 0.3μM,1μM,3μM,10μM and 30μM)and Tet(at 3μM,10μM,30μM,100μM and 150μM)decreased the I_SOCof HIRI hepatocytes;3μM Tet had statistical discrepancies compared with control(n=8,P＜0.05)and all the other groups had obvious statistical discrepancies compared with control(n=8,P＜0.01);the inhibitive effects on Isoc were concentration-dependent in HIRI hepatocytes,and the values of IC₅₀were 4.07μM and 40.20μM.Conclusion:This experiment further improved and ameliorated the method in isolating SD rat hepatocytes which settled for patch-clamp recording techniques and establishing the model of HIRI.We confirmed that the amplitude of I_SOCof hepatocytes after HIRI increased in the SD rat.This suggested that SOC was an important factor in the process of HIRI,and the incremental Ca²⁺influx through SOC was one of factors to induce the calcium overload in HIRI hepatocytes.U-73122 and Yet suppressed the increased Ca²⁺influx in a concentration-dependent way,alleviated the calcium overload in hepatocytes and protected cells on ischemia/reperfusion injury.Our research investigated the influences of SOC on HIRI-induced calcium overload in hepatocytes,and may supply theoretical and practical evidences for exploiting anti-calcium overloading drugs and resolving clinical problems.