Assessment of Respiration-Dependent Intra- and Extramitochondrial ATP Turnover: HepG2 Cancer Cells do not Utilize ATP from Oxidative Phosphorylation in the Cytosol

Abstract

BACKGROUND: Mitochondrial ATP is transported to the cytosol in exchange for ADP through the adenine nucleotide translocator (ANT). Carboxyatractyloside (CAT) and bongkrekic acid (BA) specifically inhibit ATP delivery to the cytosol via ANT, whereas oligomycin (OL) inhibits all ATP synthesis by oxidative phosphorylation. Our AIM was to assess respiration-dependent intra and extramitochondrial ATP turnover in HepG2 human hepatocarcinoma cells and cultured rat hepatocytes stimulated by ureagenic substrates. METHODS: Overnight cultured rat hepatocytes were stimulated with ureagenic substrates (in mM: 5 Na-lactate, 5 L-ornithine, 3 NH4Cl). HepG2 cells were incubated in Hank's solution or permeabilized with digitonin in intracellular buffer plus 0.5 mM ADP and 5 mM succinate. Respiration was measured using a Seahorse XF24. RESULTS: Ureagenic substrates increased respiration by hepatocytes progressively 2 to 3-fold over an hour. Subsequent addition of OL inhibited respiration to basal levels, whereas BA and CAT inhibited ureagenic respiration by ∼65%. Partial inhibition by ANT blockers was consistent with utilization of both intra- and extramitochondrial ATP in the urea cycle. In non-permeabilized HepG2 cells, OL inhibited respiration by ∼60% but BA and CAT had no effect, whereas in permeabilized HepG2 cells, OL, BA and CAT each inhibited respiration equally. In CONCLUSION, respiration inhibited by OL reflects total ATP turnover linked to oxidative phosphorylation, whereas respiration inhibited by BA or CAT reflects extramitochondrial turnover of ATP formed by oxidative phosphorylation. The difference is intramitochondrial ATP turnover. In intact HepG2 cells, ATP generated by oxidative phosphorylation was not utilized in the cytosol, although HepG2 cells contain functional ANT.