Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Abstract

Aim: Hydrogen peroxide (H2O2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods: Hepatocytes were extracted from rats. Intracellular Ca2+ concentrations ([Ca2+](i)), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results: H2O2 increased intracellular Ca2+ concentrations ([Ca2+](i)) across two kinetic phases. A low concentration (400 micromol/L) of H2O2 induced a sustained elevation of [Ca2+](i) that was reversed by removing extracellular Ca2+. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H2O2-induced membrane current increases and [Ca2+](i) elevation. A high concentration (1 mmol/L)of H2O2 induced an additional transient elevation of [Ca2+](i), which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca2+. PLC activity was increased by 1 mmol/L H2O2 but not by 400 micromol/L H2O2.

Conclusions: H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/L H2O2-induced sustained [Ca2+](i) elevation is mediated via a Ca2+ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress,reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca2+ influx.In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca2+](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca2+ stores.

Figure 1

H₂O₂ elevates [Ca²⁺]_i across two kinetic patterns in a concentration-dependent manner. In the presence of 1 mmol/L Ca²⁺ in the extracellular solution, H₂O₂ was applied to recorded hepatocytes at the concentrations of 50 μmol/L (A), 100 μmol/L (B), 400 μmol/L (C) and 1 mmol/L (D), and the effects on [Ca²⁺]_i were evaluated. The horizontal bar in each trace indicates the exposure period to H₂O₂. Representative traces are typical case from 5−12 experiments tested.

Figure 2

Effects of removal of extracellular Ca²⁺ on H₂O₂-induced elevation of [Ca²⁺]_i. (A) Under extracellular Ca²⁺-free conditions (open horizontal bar), 400 μmol/L H₂O₂ failed to induce a detectable elevation of [Ca²⁺]_i, and the reperfusion of 1 mmol/L extracellular Ca²⁺ (indicated by arrow) induced an elevation of [Ca²⁺]_i. (B) Under the same extracellular Ca²⁺-free conditions, 1 mmol/L H₂O₂ still induced a transient, initial elevation of [Ca²⁺]_i, but the sustained phase of elevation of [Ca²⁺]_i was abolished. The dashed trace represents 1 mmol/L H₂O₂-induced elevation of [Ca²⁺]_i in the presence of 1 mmol/L extracellular Ca²⁺. Representative traces in (A) and (B) are typical cases from 6−9 experiments.

Figure 3

Effects of H₂O₂ on membrane currents of hepatocytes. (A) Amphotericin B-perforated whole-cell membrane currents induced by 10 mV-increasing and decreasing voltage-step pulses before (a) and during (b) application of 400 μmol/L H₂O₂. The holding potential was -20 mV (horizontal dashed line). Ac: Current-voltage relationships obtained from the data presented in (Aa,b) before (○) and during (•) application of H₂O₂. (B) Whole-cell membrane currents recorded using the conventional whole-cell method. The pipette solution contained 5 mmol/L ATP. Bc: Current-voltage relationships obtained from the data presented in (Ba,b) before (○) and during (•) application of H₂O₂. Representative traces from 5 experiments are shown in (A) and (B). ^bP<0.05, ^cP<0.01 before application of H₂O₂, respectively.

Figure 4

ATP-sensitive conductance in hepatocytes. (A) Under perforated-patch conditions with ATP-free pipette solution, step-pulse-induced currents (a) were enhanced by conversion (suction) to conventional whole-cell configuration (b,c). (B) Under whole-cell patch conditions with ATP-free pipette solution, step-pulse-induced currents were tested at 1 (a) and 5 (b) min after conversion to whole-cell conditions and showed an enhancement of membrane currents (c). (C) Inside-out patch recordings. Currents through the membrane in response to repeatedly applied voltage-ramp pulses from -90 to 90 mV are shown (a). Initially, the concentration of ATP in the bath solution was 1 mmol/L, and then ATP was removed and an ATP-free bath solution was used. Amiloride (1 mmol/L) was applied under ATP-free conditions. Representative traces from 5 experiments are shown. The magnitudes of currents were measured at 90 mV (b) and -90 mV (c) of the transmembrane potential. Each column represents the mean from 5 series of experiments. ^bP<0.05, ^cP<0.01.

Figure 5

Effects of melatonin on H₂O₂-induced membrane current enhancement. (A) Compared to control responses (a), melatonin alone did not enhance membrane currents (b), but pre-treatment of recorded cell with 10 μmol/L melatonin for 5 min prevented 400 μmol/L H₂O₂-induced membrane current enhancement (c). After washout for 5 min, 400 μmol/L H₂O₂ induced membrane current enhancement in the same cell (d). (B) Summary of data shown in (A). Each symbol represents the mean from 6 cells tested. ^bP<0.05 vs control.

Figure 6

Effects of amiloride on H₂O₂-induced changes in membrane conductance and [Ca²⁺]_i. (A) Amphotericin B-perforated whole-cell currents recorded in the presence of 1 mmol/L amiloride before (a) and during (b) application of 400 μmol/L H₂O₂. Ac: Current-voltage relationships from the data (n=6) shown in (Aa,b) before (○) and during (•) application of H₂O₂. Ba: [Ca²⁺]_i was measured in the presence of amiloride (100 μmol/L), and then 400 μmol/L H₂O₂ was applied. Bb: The concentration of amiloride was 1 mmol/L. Bc: Normalized [Ca²⁺]_i from the data of 4−8 experiments shown in (Ba) and (Bb). Application of amiloride by itself caused a decrease in the Fura-2 intensity ratio. The change in the baseline level (Fura-2 intensity ratio) by amiloride seems to be due to some non-specific, direct reversible effect of amiloride on Fura-2 activity because both the appearance and disappearance of the effect are very quick. ^cP<0.01 vs Amiloride 0 mmol/L.

Figure 7

H₂O₂ impaired mitochondrial function. (A) Rh123 intensity changes induced by 1 mmol/L NaCN (dashed line) and 400 μmol/L H₂O₂ (bold line). (B) NAD(P)H intensity changes induced by 1 mmol/L NaCN (dashed line) and 400 μmol/L H₂O₂(bold line). (C) Summary of the effects of 1 mmol/L NaCN and 400 μmol/L H₂O₂ on NAD(P)H and Rh123. Baseline values were measured at the 2-min time point and the NaCN-induced and H₂O₂-induced responses were measured at the 4-min time point for Rh123 intensity and at the 8-min time point for NAD(P)H intensity. Representative columns from 13−33 cells tested. ^cP<0.01 vs baseline.

Figure 8

Role of the PLC pathway in H₂O₂-induced elevation of [Ca²⁺]_i. (A) Hepatocytes were pre-treated with 1 μmol/L U73122 for 30 min and then stimulated with 1 mmol/L H₂O₂ in the presence of 1 mmol/L Ca²⁺. (B) Direct measurements of PLC activity during exposure to different concentrations of H₂O₂ showed that only 1 mmol/L H₂O₂ significantly increased PLC activity. Each column represents the average from 5 experiments and the vertical bars represent SD. ^cP<0.01 vs control.