Abstract
RationaleThere is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.ObjectiveWe tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.Methods and ResultsA silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.ConclusionsSNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.
Highlights
Infants with chronic cardiopulmonary disorders associated with persistent or episodic hypoxia develop pulmonary hypertension
A silencing RNA technique was used to assess the contribution of Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) to nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling in pulmonary arterial endothelial cells (PAECs) from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline
SNAT1 silencing RNA (siRNA) inhibited the hypoxia-induced elevation in SNAT1 expression (p,0.001; Fig. 1A) and concomitantly prevented the hypoxia-induced increase in L-citrulline uptake (p = 0.01; Fig. 1B). These findings indicate that hypoxia increases L-citrulline uptake in PAECs via SNAT1 and demonstrate an association between SNAT1 and the transport of the L-arginine-NO substrate, L-citrulline, in PAECs of newborn piglets
Summary
Infants with chronic cardiopulmonary disorders associated with persistent or episodic hypoxia develop pulmonary hypertension. Impairments in nitric oxide (NO) signaling may contribute to the development of chronic hypoxia-induced pulmonary hypertension [1,2]. NO production from endothelial nitric oxide synthase (eNOS) is regulated in part by the availability of the substrate, arginine, and the cofactor, tetrahydrobiopterin (BH4) [3,4,5]. In the absence of sufficient arginine or BH4, eNOS activation generates superoxide (O2N2) instead of NO, a process known as NOS uncoupling [3,4,5]. Mechanisms that drive NOS re-coupling are poorly defined but provide potentially powerful therapeutic targets. Since L-arginine promotes eNOS coupling, strategies that effectively increase intracellular L-arginine availability to eNOS could prove beneficial. Alternate means for driving NOS re-coupling and increasing NO production merit further exploration
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