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.

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 7ß-acetoxy-8,13-epoxy-1α,6ß,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.

No effect of nitrate-rich beetroot juice on microvascular function and blood pressure in younger and older individuals: a randomised, placebo-controlled double-blind pilot study.

BACKGROUND/OBJECTIVES: To compare the effects of supplemental inorganic nitrate (NO3) on microvascular endothelial function and blood pressure in younger vs. older participants. SUBJECTS/METHODS: 25 individuals participated in a double-blind, randomised, placebo-controlled crossover pilot study. Participants were stratified by age (18-35 and ≥55 years) and consumed a single dose beetroot juice (providing 6.4 mmol NO3) or NO3-depleted beetroot juice. Blood pressure, microvascular function (via Laser Doppler Flowmetry; LDF) and urinary NO3 were assessed, and the effects of NO3 supplementation on cardiovascular parameters were compared between participants and conditions using mixed-design ANOVA. RESULTS: Treatments and methods were well tolerated, and no adverse events were reported. Urinary NO3 increased 3 h following ingestion in both groups, (P = 0.02). Levels remained elevated at 24 h post consumption in younger participants only (P = 0.02). Beetroot juice had no effect on blood pressure in either group nor on microcirculatory endothelial function. CONCLUSIONS: Beetroot juice had no effect on blood pressure or microvascular endothelial function in young and older individuals. Dosage and timing regimens for supplemental beetroot juice should be avenues for further inquiry.