The Effects Of SK&F96365 On Calcium Channels In Normal And Ischemia-Reperfusion Mice Hepatocytes

OBJECTIVE: to establish a method in isolating ICR mice hepatocytes whichsettles for patch-clamp recording techniques. Using patch-clamp techniques todetermine whether the hepatocytes plasma membrane possesses store-operated Ca²⁺channels (SOC) and subsequently study the properties of the currents of SOC (I_SOC)in normal ICR mice hepatocytes. To establish a model of hepaticischemia-reperfusion which settles for patch-clamp recording techniques, isolate theischemia-reperfusion hepatocytes, and study the properties of I_SOC inischemia-reperfusion ICR mice hepatocytes. Finally to study the effects ofSK&F96365, a known calcium channel inhibitor, on the I_SOC in normal andischemia-reperfusion ICR mice hepatocytes.METHODS: hepatocytes were obtained using ICR mice as the experimentalanimal with in situ typeâ…£collagenase perfusion. We applied whole cellpatch-clamp techniques to study SOC. In the configuration, the standard internalpipette solution contained 10mmol/ L EGTA and 2μmol/L thapsigargin to chelateCa²⁺ and prevent store refilling to active SOC. We observed the I_SOC at variousmembrane potentials. Using the method of clamping the portal vein and hepatic arteryof the median lobe and left lateral lobe for 15 minutes and reperfusion for 15 minutes,we established the model of hepatic ischemia-reperfusion which settles forpatch-clamp recording techniques, also we isolated the hepatocytes for patch-clampexperiments. 5μmol/L,10μmol/L,20μmol/L,40μmol/L,50μmol/L of SK&F96365were added to the normal and ischemia-reperfusion hepatocytes with rapidly solutionchanger to investigate the effects of SK&F96365 on I_SOC.RESULTS: the isolative method of typeâ…£collagenase perfusion in situ ismore easily with much less collagenase. The hepatocytes obtained were rounded,bright, three-dimensional with integrity structures and purity of 93%. An averageviability was 88.4%, 3.2×10⁷ hepatocytes were obtained from per liver. Afterincubation, the hepatocytes were easy to attach, form giga seal and rupture in the subsequent patch-clamp experiment. The plasma flexibility and shape of thehepatocyes could keep about 6 hours. Intracellular perfusion with EGTA andthapsigargin can decrease the concentration of Ca²⁺ in the lunmen of endoplasmicreticulum and actives SOC in the plasma that results in Ca²⁺ influx. We couldrecorded obvious I_SOC, the currents were steady and without rundown in 5 minutes.When the holding potential was 0mV and the clamping potential was-100mV, thepeak amplitude of normal hepatocytes' I_SOC was -245.95±81.45pA. The I-Vrelationship of normal hepatocytes' I_SOC generated by applying a series 200ms plulsefrom a holding potential of 0mV to different clamping potentials (-120mV to+60mV)with a 20mV increment at a frequency of 0.2Hz is linear. The reverse potential of I_SOCoccurred at voltage=0mV. The method of clamping the portal vein and hepatic arteryof the median lobe and left lateral lobe and reperfusing can successfully estabilish themodel of hepatic schemia-reperfusion with high livability of mice. Most of thehepatocytes obtained in the model were rounded, bright, three-dimensional withintegrity structures and tiny granule, but some of them had irregular shape with manyasymmetric size granule diffused in the cytoplasm. Using the microscope, we foundthat the plasma membranes of these cells were not smooth, tiny granules evenprotuberant vacuolations were on it. The average purity of the hepatocytes is 87.3%with an average viability of 79.7%. After incubation, the hepatocytes obtained fromthe model were some easy to attach, form giga seal and rupture in the subsequentpatch-clamp experiment. The plasma flexibility and shape of the hepatocyes couldkeep about 3 to 4 hours. When the holding potential was 0mV and the clampingpotential was-100mV, the peak amplitude of ischemia-reperfusion hepatocytes' I_SOCwas -443.64±53.44pA (n=15), while the control was -219.77±37.86pA (n=15), there was statistic difference between them (n=15, p＜0.01). The peak amplitudeof ischemia-reperfusion hepatocytes' I_SOC was increased, but the linear I-Vrelationship and reversal potential were not changed. In normal ICR mice hepatocytes,at the clamping potential of-100 mV, 5μmol/ L SK&F96365 decreased the peakamplitude of I_SOC from -235.19±48.19pA to -215.15±58.40pA with the inhibit rate of9.36±13.7%. The peak amplitude of I_SOC was decreased with the concentration ofSK&F96365 increasing. 50μmol/ L SK&F96365 decreased the peak amplitude of I_SOC to -39.95±7.21pA with the inhibit rate of 82.88±1.51%. All the other groups hadstatistic difference with control group except 5μmol/L SK&F96365 (P＜0.001, n=6).The amplitude of I_SOC was decreased with the concentration of SK&F96365increasing in the step command potential mode, but the linear relationship and thereverse potential were not changed. 5-50μmol/L SK&F96365 could inhibit normalmice hepatocytes' I_SOC with a concentration-dependent manner. The EC₅₀ ofSK&F96365 in this situation was 21.92μmol/L. In hepatic ischemia-reperfusion ICRmice hepatocytes, at the clamping potential of-100 mV, 5μmol/ L SK&F96365decreased the peak amplitude of I_SOC from -497.96±38.83pA to -429.03±47.80pAwith the inhibit rate of 13.43±11.54%. The peak amplitude of I_SOC was decreased withthe concentration of SK&F96365 increasing. 50μmol/L SK&F96365 decreased thepeak amplitude of Isoc to -82.24±20.47pA with the inhibit rate of 83.48±3.95%. Allthe other groups had statistic difference with control group except 5μmol/ LSK&F96365 (P＜0.001, n=9). The amplitude of Isoc was decreased with theconcentration of SK&F96365 increasing in the step command potential mode, but thelinear relationship and the reverse potential were not changed. 5-50μmol/LSK&F96365 could inhibit hepatic ischemia-reperfusion ICR mice hepatocytes' I_SOCwith a concentration-dependent manner. The EC₅₀ of SK&F96365 in this situationwas 16.99μmol/L.CONCLUSION: This experiment established a method in isolating ICR micehepatocytes which settled for patch-clamp recording techniques successfully. Weconfirmed that the ICR mice hepatocytes plasma membrane possesses SOC. Weestablished a model of hepatic ischemia-reperfusion which settled for patch-clamprecording techniques successfully, also isolated the ischemia-reperfusion hepatocytesfor studing the properties of I_SOC in this situation. The peak amplitude ofischemia-reperfusion hepatocytes' I_SOC was increased, SK&F96365 could decreasethe peak amplitude of normal and ischemia-reperfusion hepatocytes' I_SOC. It isconcluded that SOC are participated in hepatic ischemia-reperfusion injury, theampliative capacitative calcium influx through the SOC may be the improtantpathway of calcium overload in hepatic ischemia-reperfusion injury. SK&F96365 caninhibit the ampliative capacitative calcium influx and has a protective function against hepatic ischemia-reperfusion injury. All of these provide experimentalsupports for searching for new drugs against hepatocytes calcium overload andresolving the clinical difficulties.