Prolonged hypoxia augments L-citrulline transport by System A in the newborn piglet pulmonary circulation

Aims: Pulmonary arterial endothelial cells (PAECs) express the enzymes needed for generation of L-arginine from intracellular L-citrulline but do not express the enzymes needed for de novo L-citrulline synthesis. Hence, L-citrulline levels in PAECs are dependent on L-citrulline transport. Once generated, L-arginine can be converted to L-citrulline and nitric oxide (NO) by the enzyme NO synthase. We sought to determine whether hypoxia, a condition aetiologically linked to pulmonary hypertension, alters the transport of L-citrulline and the expression of the sodium-coupled neutral amino acid transporters (SNATs) in PAECs from newborn piglets. Methods and results: PAECs isolated from newborn piglets were cultured under normoxic and hypoxic conditions and used to measure SNAT1, 2, 3, and 5 protein expression and ¹⁴C-L-citrulline uptake. SNAT1 protein expression was increased, while SNAT2, SNAT3, and SNAT5 expression was unaltered in hypoxic PAECs. ¹⁴C-L-citrulline uptake was increased in hypoxic PAECs. Studies with inhibitors of System A (SNAT1/2) and System N (SNAT3/5) revealed that the increased ¹⁴C-L-citrulline uptake was largely due to System A-mediated transport. Additional studies were performed to evaluate SNAT protein expression and L-citrulline levels in lungs of piglets with chronic hypoxia-induced pulmonary hypertension and comparable age controls. Lungs from piglets raised in chronic hypoxia exhibited greater SNAT1 expression and higher L-citrulline levels than lungs from controls. Conclusion: Increased SNAT1 expression and the concomitant enhanced ability to transport L-citrulline in PAECs could represent an important regulatory mechanism to counteract NO signalling impairments known to occur during the development of chronic hypoxia-induced pulmonary hypertension in newborns.