L‐citrulline increases arginase II protein levels and arginase activity in hypoxic piglet pulmonary artery endothelial cells

Abstract

The L-arginine precursor, L-citrulline, re-couples endothelial nitric oxide synthase (eNOS), increases nitric oxide (NO) production, and ameliorates chronic hypoxia-induced pulmonary hypertension (PH) in newborn pigs. L-arginine can induce arginase, which, in turn, may diminish NO production. Our major purpose was to determine if L-citrulline increases arginase activity in hypoxic piglet pulmonary arterial endothelial cells (PAECs), and if so, concomitantly impacts the ability to increase eNOS re-coupling and NO production. Piglet PAECs were cultured in hypoxic conditions with L-citrulline (0-3 mM) and/or the arginase inhibitor S-(2-boronoethyl)-L-cysteine, BEC. We measured arginase activity and NO production. We assessed eNOS coupling by measuring eNOS dimers and monomers. L-citrulline concentrations > 0.5 mM increased arginase activity in hypoxic PAECs. L-citrulline concentrations > 0.1 mM increased NO production and concentrations > 0.5 mM elevated eNOS dimer-to-monomer ratios. Co-treatment with L-citrulline and BEC elevated eNOS dimer-to-monomer ratios more than sole treatment. Despite inducing arginase, L-citrulline increased NO production and eNOS coupling in hypoxic piglet PAECs. However, these dose-dependent findings raise the possibility that there could be L-citrulline concentrations that elevate arginase to levels that negate improvements in eNOS dysfunction. Moreover, our findings suggest that combining an arginase inhibitor with L-citrulline merits evaluation as a treatment for chronic hypoxia-induced PH.

Folic acid, either solely or combined with L‐citrulline, improves NO signaling and ameliorates chronic hypoxia‐induced pulmonary hypertension in newborn pigs

“…However, we were concerned with the possibility of uncovering adverse events from using higher L‐citrulline doses. One possible adverse consequence, raised by our recent in vitro findings (Douglass et al, 2021 ), is that progressively increasing the dose of L‐citrulline would elevate arginase activity to levels adversely impacting NO signaling. We were also mindful that newborns with chronic lung and heart diseases sometimes develop pulmonary edema when given high fluid volumes.…”

“…It is possible that higher doses of L‐citrulline might further reduce PVR and enhance NO production in vivo in hypoxic piglets. However, we recently performed in vitro studies and found that L‐citrulline dose‐dependently increases arginase activity in hypoxic piglet pulmonary artery endothelial cells (Douglass et al, 2021 ). This latter finding is concerning because arginase can decrease NO production by competing with eNOS for the substrate, L‐arginine (Akashi et al, 2001 ; Morris, 2009 ).…”

Impact of l‐citrulline on nitric oxide signaling and arginase activity in hypoxic human pulmonary artery endothelial cells

“…For arginase I and II and eNOS analysis, using previously described methods, aliquots of supernatant were applied to tris‐glycine precast 4%–20% polyacrylamide gels so that equal amounts of protein were loaded. 16 After electrophoresis at 120 volts for 2 h, the protein was transferred to a polyvinylidene difluoride membrane. Nonspecific protein binding was blocked by incubating the membrane at room temperature in PBS and 0.1% Tween‐20 (PBS‐T; Sigma‐Aldrich) containing 5% nonfat dried milk and then washing in PBS‐T containing 1% nonfat dried milk.…”

Investigation of the ubiquitin proteasome system in pulmonary arterial hypertension

“…In addition, the loss of the mitochondrial uncoupled protein (UCP2) in endothelial cells caused increased PINK1 and Parkin 49 . Arginase II protein (ArgII) regulates Parkin-dependent p32 degradation and it contributes to eNOS activation 50 . USP30 is an OMM-localized deubiquitinase that antagonizes PINK1/Parkin-driven mitophagy in mammalian cells by deubiquitinating the OMM substrate.…”