Dysregulation of L-arginine metabolism and bioavailability associated to free plasma heme

Abstract

Severe Plasmodium falciparum malaria is associated with hypoargininemia, which contributes to impaired systemic and pulmonary nitric oxide (NO) production and endothelial dysfunction. Since intravascular hemolysis is an intrinsic feature of severe malaria, we investigated whether and by which mechanisms free heme [Fe(III)-protoporphyrin IX (FP)] might contribute to the dysregulation of L-arginine (L-Arg) metabolism and bioavailability. Carrier systems "y+" [or cationic amino acid transporter (CAT)] and "y+L" transport L-Arg into red blood cells (RBC), where it is hydrolyzed to ornithine and urea by arginase (isoform I) or converted to NO* and citrulline by endothelial nitric oxide synthase (eNOS). Our results show a significant and dose-dependent impairment of L-Arg transport into RBC pretreated with FP, with a strong inhibition of the system carrier y+L. Despite the impaired L-Arg influx, higher amounts of L-Arg-derived urea are produced by RBC preexposed to FP caused by activation of RBC arginase I. This activation appeared not to be mediated by oxidative modifications of the enzyme. We conclude that L-Arg transport across RBC membrane is impaired and arginase-mediated L-Arg consumption enhanced by free heme. This could contribute to reduced NO production in severe malaria.

Fig. 1.

A and C: Ultraviolet-visible (UV/Vis) spectra of 10 mM ferriprotoporphyrin IX (FP) alone (solid line) and in the presence of 250 mM l-arginine (l-Arg) (dashed line; A) or 250 mM l-lysine (l-Lys) (dashed line; C). B: UV/Vis spectra of 10 mM hematoporphyrin IX (HP) alone (solid line) and in the presence of 250 mM l-Arg (dashed line). Abs, absorbance.

Fig. 2.

Eadie-Hofstee graph for arginase activity of control (C) and 40 μM FP-pretreated (FP) red blood cells (RBC). Each data point represents a triplicate sample of 4 separate experiments. V₀, initial velocity.

Fig. 3.

A: urea production in total lysates of control RBC or RBC pretreated with 40 μM FP (60 min, 37°C). Incubation was performed for 16 h at 37°C in the presence of increasing concentrations of l-Arg (0.1–5 mM). This is a representative experiment out of 5, made in triplicate. B: mean urea values from 5 paired lysates, cytosols, and ghosts prepared from control and FP samples (40 μM FP, 60 min, 37°C). Incubation was performed for 16 h at 37°C in the presence of 5 mM l-Arg. Paired comparisons were made by Student's t-test.

Fig. 4.

A: urea released in the extracellular medium by control RBC or RBC pretreated for 60 min with increasing concentrations of FP and then incubated for 16 h at 37°C in the presence of 5 mM l-Arg. Results are expressed as absolute values (mM) or per milligram of Hb. Results are means ± SD of 4 experiments. *P < 0.01 vs. control. B: urea released in the extracellular medium (n = 5 experiments) by control RBC, RBC pretreated for 60 min with 40 μM FP or HP, or RBC pretreated for 3 hrs with Fe=ascorbate (Fe-Asc, 0.3–0.5 mM). CO-FP indicates that the RBC suspension was exposed for 10 min to a carbon monoxide atmosphere before treatment with 40 μM FP. Bars represent means and 95% confidence intervals (CI). Comparison over all groups: P < 0.001 (4 df); paired comparisons with control: *P = 0.04 by Wilcoxon signed-rank test.

Fig. 5.

A: total influx of l-[³H]Arg (200 μM) in control RBC or RBC pretreated with increasing concentrations of FP. Influx of l-Arg was determined over 5-min incubation. Bars represent mean (5 experiments) and 95% CI. Test for trend: P < 0.001. B: time course of total l-[³H]Arg (200 μM) influx in control RBC or RBC pretreated with 40 μM FP. Results are means ± SD of 4 experiments.

Fig. 6.

A: l-[³H]Arg uptake into control RBC or RBC pretreated with 40 μM FP (n = 4 experiments/treatment). Rates were measured in the absence (no addition) or the presence of either 1 mM unlabeled leucine (Leu) or 2 mM N-ethylmaleimide (NEM). Boxes indicate medians and interquartile ranges. Bars represent upper and lower adjacent values. P = 0.068 for each paired comparison between treatment and control by Wilcoxon signed-rank test. B: contribution of transport systems y+L and y+ and free diffusion to the total l-[³H]Arg transport in control RBC or RBC pretreated with 40 μM FP.