The role of TRPM2 channels in oxidative stress-induced liver damage.

Abstract

The increased production of highly reactive oxygen and nitrogen species plays a significant role in development of a number of liver disorders associated with hepatocellular death and impaired cell regeneration. Liver injury induced by drug toxicity, ischemia-reperfusion, excessive alcohol consumption and different types of viral hepatitis is in large part mediated by oxidative stress. Liver damage due to oxidative stress induced by drugs, including acetaminophen, accounts for 5% of all hospital admissions and for almost half of all acute liver failures. One of the features of hepatocellular death mediated by oxidative stress is Ca²⁺ overload due its release from intracellular organelles and activation of ion channels on the plasma membrane. Ca²⁺ is fundamental for normal cellular functioning. Ca²⁺ signalling, mediated by the rise in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]c)[c subscript], regulates many cellular events. However, a sustained rise in [Ca²⁺]c [c subscript] can be detrimental, leading to mitochondrial dysfunction and cell death through apoptosis and necrosis. Although it is well recognised that Ca²⁺ plays a significant role in oxidative stress-induced liver damage, the molecular identities of the ion channels that provide a pathway for Ca²⁺ entry in hepatocytes remain unidentified. One of the potential candidates that could be responsible for such Ca²⁺ entry pathway in hepatocytes is Transient Receptor Potential Melastatin 2 (TRPM2) channel. TRPM2 is a non-selective cation channel permeable to Na⁺ and Ca²⁺. The main physiological activator of TRPM2 channel is ADP-ribose, which binding to NUDT9-H motif in the TRPM2 C-terminus leads to the opening of the channel pore. It is known that oxidative stress promotes generation and release of ADPR from mitochondria and nuclei into the cytoplasmic space, thus promoting activation of TRPM2-mediated Ca²⁺ entry. In this thesis, we hypothesised that oxidative stress-induced Ca²⁺ entry in hepatocytes is mediated by TRPM2 channels, and used acetaminophen overdose as a model of oxidative stress-induced liver damage. We show that hepatocytes express long isoform of TRPM2, which mediates ADPR- and H₂O₂-induced Ca²⁺ entry and the cation current in these cells. Furthermore, we show that TRPM2 channels are activated in hepatocytes treated with high concentrations of acetaminophen and are responsible for Ca²⁺ overload in acetaminophen-induced liver toxicity. Experiments using TRPM2 KO mice provide first evidence of a pivotal role of TRPM2 channels in acetaminophen-induced liver injury, showing that lack of TRPM2 expression largely protects liver from acetaminophen overdose. An important finding that TRPM2 channels translocate from intracellular compartments to the plasma membrane provides explanation for a slow development of Ca²⁺ entry in response to H₂O₂ and acetaminophen. Finally, we show that substances previously known to protect liver from acetaminophen-induced damage are, in fact, inhibitors of TRPM2 current. Chlorpromazine, an antipsychotic drug, reversibly blocks TRPM2 channel pore, and curcumin, a chemical found in common spice, potently blocks activation of TRPM2 current by ADPR. The results presented in this thesis provide a fundamental knowledge about the role of TRPM2 channels in oxidative stress-induced liver injury, but also open a new chapter in search for the new drugs and drug targets for the treatment of a number of oxidative stress-related liver pathologies.