Mechanisms of oxygen radicals-induced cell injury in rat hepatocytes. -The study with intracellular ATP transduction.

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14570035
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General physiology
• Research Institution: Hirosaki University
• Principal Investigator: TAKEO Teruko Hirosaki University School of Medicine, Associate Professor, 医学部, 助教授 (20113813)
• Co-Investigator(Kenkyū-buntansha): WAKUI Makoto Hirosaki University School of Medicine, Professor, 医学部, 教授 (80108505)
• Project Period (FY): 2002 – 2003
• Keywords: hydrogen-peroxide / hepatocyte / calcium dynamics / phospholipase C / intracellular ATP / nonselective cation channel

Research Abstract

Development of ischemic-reperfusion injury is a major risk factor of liver transplantation. One of the mechanisms by which ischemic-reperfusion injury occurs is oxidative stress to liver cells (hepatocytes), occurring when pro-oxidants overwhelm cellular antioxidant defense mechanisms. In this oxidation process, an increase in intracellular Ca^<2+> concentration ([Ca^<2+>]i) is known to facilitate progression of cellular damage. In the present study, we investigated the effect of hydrogen peroxide (H_2O_2) on the Ca^<2+> dynamics of freshly isolated rat hepatocytes. And the following results were obtained.
1.H_2O_2 increased the level of [Ca^<2+>]_i of hepatocytes by mediating Ca^<2+> entry from extracellular fluid.
2.The membrane conductance of the hepatocytes was not acutely changed by H_2O_2 application. However, after several minutes exposure to H_2O_2, it gradually increased.
3.A removal of intracellular ATP by using a pipette solution with null ATP increased the membrane conductance via nonselective cation channels.
4.H_2O_2 reduced the ATP content in hepatocytes.
5.H_2O_2 augmented Ca^<2+> oscillations induced by phenylephrine through α_1-adrenoceptors, and 2APB inhibited both the phenylephrine and H_2O_2 effects.
6.H_2O_2 increased the activity of phospholipase C (PLC).
7.Amiloride (nonselective cation channel inhibitor) and U73122 (PLC inhibitor) prevented the H_2O_2-induced elevation of [Ca^<2+>]i.
These results suggest that in rat hepatocytes, H_2O_2 induced intracellular Ca^<2+> mobilization, which was closely related with PLC activity and nonselective cation channels. At present, we have no evidence to support or refute the possibility that H_2O_2 affects plasma membrane Ca^<2+> pump or Ca^<2+>/Na^+ exchange system.