Diltiazem Protects Hepatocytes from Damage Induced by Reactive Oxygen Species Through Actions on Mitochondria

Abstract

ABSTRACT

AIM: d(+)-cis-Diltiazem, in addition to its ability to inhibit voltage-operated Ca²⁺ channels, exhibits antioxidant properties and can protect hepatocytes in the diseased liver.  However, the mechanisms involved are not well understood. The aims were to determine whether the protective effects of diltiazem on damaged hepatocytes include actions on the mitochondrial Ca²⁺ channels that enable damaged cells to continue to produce energy.

MATERIALS AND METHODS: Rates of O₂ consumption by isolated rat liver mitochondria were measured using an O₂ electrode. Isolated rat hepatocytes or H4IIE rat liver cells were incubated with H₂O₂. Cell damage was assessed by measuring extracellular lactate dehydrogenase spectrophotometrically.

RESULTS: In isolated mitochondria, Ca²⁺ (170 µM) added to partly uncouple oxidative phosphorylation decreased the acceptor control ratio from 3.5 ± 0.1 to 1.9 ± 0.1.  d(+)-cis-Diltiazem (410 µM) pre-incubated for 5 min increased the acceptor control ratio, measured in the presence of Ca²⁺, to 2.6 ± 0.1 (n = 18-31, P ≤ 0.001).  Co-addition of thiamine did not affect the actions of diltiazem. About 50% of the added diltiazem was associated with the mitochondrial membranes. Incubation of hepatocytes with H₂O₂ for 24 h induced substantial cell injury.  Pre-incubation with d(+)-cis-diltiazem (5 µM) for 2 h reduced this by about 50%.  Some additional protection was given by co-addition of thiamine (50 µM).  Protection provided by d(+)-cis-diltiazem was comparable with that provided by silymarin.

CONCLUSION: Diltiazem protects hepatocytes from damage induced by reactive oxygen species.  The mechanism may involve, in part, antioxidant actions of diltiazem on mitochondrial and other intracellular membranes.