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

Impaired nitric oxide (NO) signaling contributes to the development of pulmonary hypertension (PH). The l-arginine precursor, l-citrulline, improves NO signaling and has therapeutic potential in PH. However, there is evidence that l-citrulline might increase arginase activity, which in turn, has been shown to contribute to PH.

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

Concomitant with developing pulmonary hypertension (PH), newborn piglets exposed to chronic hypoxia develop pulmonary vascular NO signaling impairments. PH is reduced and NO signaling is improved in chronically hypoxic piglets treated with the NO-arginine precursor, L-citrulline. Folic acid positively impacts NO signaling.

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

The L-arginine precursor, L-citrulline, re-couples endothelial nitric oxide synthase, increases nitric oxide production, and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs. L-arginine can induce arginase, which, in turn, may diminish nitric oxide production. Our major purpose was to determine if L-citrulline increases arginase activity in hypoxic piglet pulmonary arterial endothelial cells, and if so, concomitantly impacts the ability to increase endothelial nitric oxide synthase re-coupling and nitric oxide production.

l-Citrulline treatment alters the structure of the pulmonary circulation in hypoxic newborn pigs.

Background: Dysregulated nitric oxide (NO) signaling contributes to chronic hypoxia (CH)-induced pulmonary hypertension (PH). NO signaling is improved and pulmonary vascular resistance (PVR) is reduced in CH piglets treated with the l-arginine-NO precursor, l-citrulline. We hypothesized that l-citrulline might cause structural changes in the pulmonary circulation that would contribute to the reduction in PVR and that the l-citrulline-induced structural changes would be accompanied by alterations in vascular endothelial growth factor (VEGF) signaling.

Combined l-citrulline and tetrahydrobiopterin therapy improves NO signaling and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Newborn pigs with chronic hypoxia-induced pulmonary hypertension (PH) have evidence of endothelial nitric oxide synthase (eNOS) uncoupling. In this model, we showed that therapies that promote eNOS coupling, either tetrahydrobiopterin (BH), a NOS cofactor, or l-citrulline, a NO-l-arginine precursor, inhibit PH. We wanted to determine whether cotreatment with l-citrulline and a BH compound, sapropterin dihydrochloride, improves NO signaling and chronic hypoxia-induced PH more markedly than either alone.

Reactive oxygen species modulate Na-coupled neutral amino acid transporter 1 expression in piglet pulmonary arterial endothelial cells.

We have previously shown that Na-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in pulmonary arterial endothelial cells (PAECs) from newborn piglets. Specifically, the ability to increase NO production in response to the l-arginine-NO precursor l-citrulline is dependent on SNAT1 expression. Elucidating factors that regulate SNAT1 expression in PAECs could provide new insights and therapeutic targets relevant to NO production.

A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a deadly disease with no cure. Alternate conversion of angiotensin II (AngII) to angiotensin-(1-7) (Ang-(1-7)) by angiotensin-converting enzyme 2 (ACE2) resulting in Mas receptor (Mas1) activation improves rodent models of PAH. Effects of recombinant human (rh) ACE2 in human PAH are unknown.

Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

We previously showed that newborn piglets who develop pulmonary hypertension during exposure to chronic hypoxia have diminished pulmonary vascular nitric oxide (NO) production and evidence of endothelial NO synthase (eNOS) uncoupling (Fike CD, Dikalova A, Kaplowitz MR, Cunningham G, Summar M, Aschner JL. Am J Respir Cell Mol Biol 53: 255-264, 2015). Tetrahydrobiopterin (BH) is a cofactor that promotes eNOS coupling.

Rescue Treatment with L-Citrulline Inhibits Hypoxia-Induced Pulmonary Hypertension in Newborn Pigs.

Infants with cardiopulmonary disorders associated with hypoxia develop pulmonary hypertension. We previously showed that initiation of oral L-citrulline before and continued throughout hypoxic exposure improves nitric oxide (NO) production and ameliorates pulmonary hypertension in newborn piglets. Rescue treatments, initiated after the onset of pulmonary hypertension, better approximate clinical strategies.

Reactive oxygen species scavengers improve voltage-gated K(+) channel function in pulmonary arteries of newborn pigs with progressive hypoxia-induced pulmonary hypertension.

Abstract Changes in voltage-gated K(+) (Kv) channel function contribute to the pathogenesis of pulmonary hypertension. Yet the mechanisms underlying Kv channel impairments in the pulmonary circulation remain unclear. We tested the hypothesis that reactive oxygen species (ROSs) contribute to the Kv channel dysfunction that develops in resistance-level pulmonary arteries (PRAs) of piglets exposed to chronic in vivo hypoxia.

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.

Effect of a phosphodiesterase 5 inhibitor on pulmonary and cerebral arteries of newborn piglets with chronic hypoxia-induced pulmonary hypertension.

Background: The use of phosphodiesterase 5 (PDE5) inhibitors to treat newborns with pulmonary hypertension is increasing. The effect of PDE5 inhibitors on the neonatal cerebral circulation remains unknown. The neonatal piglet model of chronic hypoxia-induced pulmonary hypertension allows the study of the effects of PDE5 inhibitors on both the pulmonary and cerebral circulations.

Pulmonary arterial responses to reactive oxygen species are altered in newborn piglets with chronic hypoxia-induced pulmonary hypertension.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of pulmonary hypertension. ROS might mediate vascular responses, at least in part, by stimulating prostanoid production. Our goals were to determine whether the effect of ROS on vascular tone is altered in resistance pulmonary arteries (PRAs) of newborn piglets with chronic hypoxia-induced pulmonary hypertension and the role, if any, of prostanoids in ROS-mediated responses.

Protein complex formation with heat shock protein 90 in chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Aberrant interactions between heat shock protein (Hsp)90 and its client proteins could contribute to pulmonary hypertension. We tested the hypotheses that 1) the interaction between Hsp90 and its known client protein, endothelial nitric oxide synthase (eNOS), is impaired in pulmonary resistance arteries (PRAs) from piglets with pulmonary hypertension caused by exposure to 3 or 10 days of hypoxia and 2) Hsp90 interacts with the prostanoid pathway proteins prostacyclin synthase (PGIS) and/or thromboxane synthase (TXAS). We also determined whether Hsp90 antagonism with geldanamycin alters the agonist-induced synthesis of prostacyclin and thromboxane or alters PRA responses to these prostaglandin metabolites.

L-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Newborn piglets develop pulmonary hypertension and have diminished pulmonary vascular nitric oxide (NO) production when exposed to chronic hypoxia. NO is produced by endothelial NO synthase (eNOS) in the pulmonary vascular endothelium using l-arginine as a substrate and producing l-citrulline as a byproduct. l-Citrulline is metabolized to l-arginine by two enzymes that are colocated with eNOS in pulmonary vascular endothelial cells.

Heat shock protein 90-eNOS interactions mature with postnatal age in the pulmonary circulation of the piglet.

Binding of endothelial nitric oxide synthase (eNOS) to the chaperone protein, Hsp90, promotes coupled eNOS synthetic activity. Using resistance level pulmonary arteries (PRA) from 2-day-, 5- to 7-day-, and 12-day-old piglets, we tested the hypothesis that Hsp90-eNOS interactions are developmentally regulated in the early neonatal period. PRA were isolated for coimmunoprecipitation and immunoblot analyses or cannulated for continuous diameter measurements using the pressurized myography technique.

Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension.

Our main objective was to determine whether reactive oxygen species (ROS), such as superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)), contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the cell-permeable superoxide dismutase mimetic (SOD; M40403) and/or PEG-catalase (PEG-CAT) on responses to acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary resistance arteries (PRAs; 90-to-300-microm diameter).

Heat shock protein 90 modulates endothelial nitric oxide synthase activity and vascular reactivity in the newborn piglet pulmonary circulation.

Heat shock protein 90 (Hsp90) binding to endothelial nitric oxide synthase (eNOS) is an important step in eNOS activation. The conformational state of bound Hsp90 determines whether eNOS produces nitric oxide (NO) or superoxide (O(2)(*-)). We determined the effects of the Hsp90 antagonists geldanamycin (GA) and radicicol (RA) on basal and ACh-stimulated changes in vessel diameter, cGMP production, and Hsp90:eNOS coimmunoprecipitation in piglet resistance level pulmonary arteries (PRA).

Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Our purpose was to determine whether smooth muscle cell membrane properties are altered in small pulmonary arteries (SPA) of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A microelectrode technique was used to measure smooth muscle cell membrane potential (E(m)) in cannulated, pressurized SPA (100- to 300-microm diameter).

Sildenafil and an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Devising therapies that might prevent the onset or progression of pulmonary hypertension in newborns has received little attention. Our major objective was to determine whether sildenafil, a selective phosphodiesterase inhibitor, prevents the development of an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs. Another objective was to determine whether sildenafil causes pulmonary vasodilation without systemic vasodilation in piglets with chronic pulmonary hypertension.

Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.

The pulmonary vasoconstrictor, thromboxane, may contribute to the development of pulmonary hypertension. Our objective was to determine whether a combined thromboxane synthase inhibitor-receptor antagonist, terbogrel, prevents pulmonary hypertension and the development of aberrant pulmonary arterial responses in newborn piglets exposed to 3 days of hypoxia. Piglets were maintained in room air (control) or 11% O(2) (hypoxic) for 3 days.

Cyclooxygenase-2 and an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Our objective was to determine whether cyclooxygenase (COX)-2-dependent metabolites contribute to the altered pulmonary vascular responses that manifest in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the COX-2 selective inhibitor NS-398 on responses to arachidonic acid or acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary arteries (100- to 400-microm diameter).

Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets.

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O(2)) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, N(omega)-nitro-L-arginine methylester (L-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured.

Exhaled NO is reduced at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets.

Altered nitric oxide (NO) production could contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. To determine whether parameters of lung NO are altered at an early stage of hypoxia-induced pulmonary hypertension, newborn piglets were exposed to room air (control, n = 21) or 10% O(2) (hypoxia, n = 19) for 3-4 days. Some lungs were isolated and perfused for measurement of exhaled NO output and the perfusate accumulation of nitrite and nitrate (NOx-), the stable metabolites of NO.