Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors
| Autor: | Manon Buist-Homan, Martina Schmidt, Xiaodong Cheng, Nshunge Musheshe, Han Moshage, Asmaa Oun, Frank Lezoualc'h, Fabio Alejandro Aguilar Mora |
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| Přispěvatelé: | Molecular Pharmacology, Groningen Research Institute for Asthma and COPD (GRIAC), Restoring Organ Function by Means of Regenerative Medicine (REGENERATE), Center for Liver, Digestive and Metabolic Diseases (CLDM) |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Male EXPRESSION Programmed cell death IBMX Diclofenac INHIBITION Pharmacology Mitochondrion MECHANISMS 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine medicine Cyclic AMP MITOCHONDRIAL TOXICITY KINASE Animals Guanine Nucleotide Exchange Factors Humans CELL Rats Wistar Protein kinase A Liver injury Forskolin INDUCED LIVER-INJURY EPAC Anti-Inflammatory Agents Non-Steroidal medicine.disease Rats APOPTOSIS 030104 developmental biology HEK293 Cells chemistry Apoptosis Hepatocytes Molecular Medicine Signal transduction 030217 neurology & neurosurgery |
| Zdroj: | Molecular Pharmacology, 99(4), 294-307. AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS |
| ISSN: | 0026-895x |
| Popis: | Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4) and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction. |
| Databáze: | OpenAIRE |
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