Effects Of Agonists/Blockers Of Store-operated Ca²⁺ Channels On The Calcium Oscillations In Normal Hepatocytes And Hepatocytes Subjected To Ischemia And Reperfusion (I/R) Injury

AIM:To establish a method in isolating normal hepatocytes and hepatocytes subjected to ischemia and reperfusion(I/R)injury,which settles for calcium imaging techniques.To investigate the role of SOCs(store-operated channels)in the agonists(noradrenali,ATP,vasopressin)-induced oscillations of the cytoplasmic free Ca²⁺concentration([Ca²⁺]_cyt)in normal hepatocytes and hepatocytes subjected to ischemia and reperfusion(I/R)injury.To investigate the effects of inhibitors of SOCs(tetrandrine,U73122,niflumic acid)in normal hepatocytes and hepatocytes subjected to ischemia and reperfusion(I/R)injury.To investigate the effects of arachidonic acid(agonist of non-selective cation channels(NSCCs))on the frequency of normal hepatocytes.METHODS:Liver cells were isolated by in situ perfusion via the portal vein with Ca²⁺/ Mg²⁺-free Krebs buffer followed by collagenase digestion from normal Sprague Dawley rats or rats subjected to hepatic I/R injury.Ca²⁺oscillations induced by noradrenalin,ATP and vasopressin were measured by means of the Ca²⁺-sensitive dye Fluo-4-AM,recorded by calcium imaging techniques.The inhibitors of SOCs(tetrandrine,U73122 and niflumic acid)were added after oscillations induced by agonists(noradrenali,ATP,vasopressin)of SOCs.To investigate the effect of arachidonic acid(agonist of non-selective cation channels (NSCCs))on the frequency of calcium oscillations in normal cells.RESULTS:Liver cells were obtained with the purity of 85%and an average viability of 85%.Noradrenalin(100 nM),ATP(1.2μM),and vasopressin(0.5 nM)evoked Ca²⁺oscillation in the normal hepatocytes.However,concentrations of these agonists to evoke Ca²⁺oscillation were much lower in hepatocytes isolated from livers subjected to I/R injury than that in the normal hepatocytes(noradrenalin 10 nM, ATP 0.6μM,and vasopressin 0.1nM).Tetrandrine(100μM),U73122(25μM),and niflumic acid(20μM)completely blocked Ca²⁺oscillations induced by noradrenalin, ATP,and vasopressin in the normal and I / R injured hepatocytes and the block was rapid and reversible.Tetrandrine induced a concentration-dependent decrease of the frequency of Ca²⁺oscillation in normal hepatocytes and hepatocytes subjected to ischemia and reperfusion(I/R)injury.The IC₅₀value of noradrenalin(100nM) induced calcium oscillations in normal hepatocytes was 31.754μM while in hepatocytes subjected to ischemia and reperfusion(I / R)injury was 45.994μM.The IC₅₀value of noradrenalin(10nM)induced calcium oscillation in hepatocytes subjected to ischemia and reperfusion(I / R)injury was 26.458μM.CONCLUSION:This experiment established a method in isolating normal liver cells and liver cells subjected to ischemia and reperfusion(I / R)injury which settled for calcium imaging techniques successfully.Noradrenalin,ATP,and vasopressin are agonists of SOCs and they can evoke oscillations both in normal hepatocytes and hepatocytes subjected to ischemia and reperfusion(I / R)injury.In addition,our data indicate that Ca²⁺inflow through SOCs is required for the maintenance of Ca²⁺ oscillations in the normal and I / R injured hepatocytes.Arachidonic acid does not activate NSCCs,but does inhibit calcium oscillations.These results indicate that NSCCs do not contribute to maintain calcium oscillations in liver cells.Our data demonstrate that I / R injury sensitizes hepatocytes to Ca²⁺oscillations,which could result in hepatocyte damage.Tetrandrine,U73122,niflumic acid might be effective blockers of SOCs.