Cystathionine Gamma‐Lyase Inhibition in Primary Human Hepatocytes Affects the Production of Hydrogen Sulfide Biomarkers through the Transsulfuration Pathway

Abstract

Hydrogen sulfide (H2S) has been identified as a neurotransmitter involved in smooth muscle dilation and thus, may have cardioprotective effects in‐vivo. The transsulfuration enzymes, cystathionine beta‐synthase (CBS) and cystathionine gamma‐lyase (CSE), produce the majority of endogenous H2S concurrent with the formation of lanthionine (lan) and homolanthionine (hlan) biomarkers. We compared transsulfuration pathway kinetics and H2S biomarkers in primary human hepatocytes with and without functionality of enzyme CSE. CSE was inhibited by 1000 µM propargylglycine (PAG) and metabolic fluxes were determined using [U‐13C5] L‐methionine and (3,3 D2) L‐cysteine precursors. Methionine cycle kinetics and rate of homocysteine production were not affected by the addition of PAG. In the presence of PAG, cystathionine was accumulated in cells and media (P<0.001). Intracellular hlan concentration and fractional synthesis rates (FSR) were higher in untreated cells compared to cells with CSE inhibited (P<0.041), while in media hlan concentration was 50% less due to PAG inhibition (P<0.025). In the presence of PAG, lan concentration in cells and media exceeded those of untreated cells (P<0.012). The inhibition of CSE in primary hepatocytes greatly decreased the production of H2S biomarker hlan, while lan production was not affected. These results support in‐vitro studies indicating that CSE is the primary enzyme in hlan production while CBS produces the majority of lan. (Supported by NIH DK072398 and CIHR MOP133505).