Requirement of argininosuccinate lyase for systemic nitric oxide production

Abstract

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Figures

Figure 1. Physiological phenotype of AslNeo/Neo mice

(a) Growth and hair phenotype. (b) Kaplan Meier survival curve for AslNeo/Neo mice (n=22; solid line) vs. WT mice (n=19; dotted line) (*p<0.002). (c) Weight for AslNeo/Neo mice (n=5) vs. WT (n=4) or heterozygous (n=6) littermates. WT: wild type; AslNeo/+: heterozygous mice; AslNeo/Neo: homozygous mice (*p<0.05). (d) Renal Function for AslNeo/Neo mice (n=4) vs. WT littermates (n=5) (*p<0.005). (e) Liver transanimases for AslNeo/Neo mice (*p<0.05). (f) Blood pressure measurements for AslNeo/Neo mice (n=6) vs. WT littermates (n=4) (*p<0.0002). (g) Blood pressure in Nos3+/−;AslNeo/+ (n=9) double heterozygous vs. Nos3+/−;Asl+/+ mice (n=4) (**p<2×10−8).

Figure 2. Decreased NO production with ASL deficiency

(a,b) Nitric oxide status in AslNeo/Neo mice (n=3) and WT littermates (n=5). (a) Nitrosothiol measurements (RSNO) (*p<0.05). (b) Nitrite measurements (*p<0.05). (c) Representative Western Blot analyses of lung tissue samples from 10 AslNeo/Neo mice and 10 WT control. Antibodies used are labeled on side. Protein densitometry is shown in the bottom panel.

Figure 3. Pharmacological rescue with an NO source

(a) Kaplan Meier survival curves comparing different treatment modalities vs. placebo in AslNeo/Neo mice (*p<0.007) (n = 15–22 in each group). (b) Kaplan-Meier survival analysis (*p<0.001 on log ranks) between the arginine plus sodium benzoate treated group (n=16), the sodium nitrite treated group (n=27) and the triple therapy group (n=22), when compared to the placebo group (n=22). (c) Left panel: Weight gain with sodium nitrite treatment (n=29) and the triple therapy treatment (n=22) as compared to placebo (n=26) and to the standard treatment with arginine and sodium benzoate (n=17) group (one-way ANOVA, *p <0.05). Right panel: A representative picture of WT control and AslNeo/Neo mice at 8 weeks of age after receiving the triple therapy. (d) Nitrosothiols (RSNO) were measured in liver of AslNeo/Neo and WT mice treated with placebo vs. triple therapy (arginine, sodium nitrite and sodium benzoate) (*p<0.01; **p<0.009). (e) Blood pressure measurements in WT (n=3) vs. AslNeo/Neo (n=4) on triple therapy with sodium benzoate, arginine, and sodium nitrite. (f,g) Aortic ring relaxation. (f) A representative graph for the difference in WT aortic rings relaxation vs. AslNeo/Neo aortic rings relaxation (p<0.004). The graph represents the percentage relaxation in response to acetylcholine measured in 3 WT and 3 AslNeo/Neo aortae, 4 segments from each aorta were analyzed (p<0.004). (g) Representative tracing from WT vs. AslNeo/Neo aortic ring isometric tension studies showing percentage relaxation in response to acetylcholine (Ach), arginine (Arg), and sodium nitroprusside (SNP) treatment

Figure 4. Asl knockdown in piglet endothelial cells decreases nitrite production in response to arginine

(a) Relative RNA expression in pooled RNA from primary piglet pulmonary artery (PA) endothelial cells transfected with Asl siRNA (ASL-RNAi) compared to non-specific siRNA (NS-RNAi). Endothelial specific von Willebrand factor (vWF) and NOS3 levels are shown as control. (b) Western blot using antibody to ASL in endothelial cells transfected with ASL siRNA as compared to NS-RNAi siRNA. The densitometric quantification is below. (c) Measurement of the relative nitrite levels in the media of piglet PA endothelial cells transfected with ASL siRNA vs. NS siRNA in response to Bradykinin (10 µM) treatment (for 24 hours) with and without the NOS inhibitor L-NAME (100 µM) (*p<0.05). The experiment was performed in triplicate. Please refer to the online methods for the specific calculation (d) Measurement of the relative nitrite levels in the media of piglet PA endothelial cells transfected with ASL siRNA vs. NS siRNA in response to L-Arginine (10 mM) treatment (for 60 minutes) with and without L-NMMA (500 µM) or D-Arginine as control (*p< 0.01, **p<0.005). The experiment was performed in triplicates. NS: Non-significant.

Figure 5. ASL deficient human cells and patients do not efficiently generate NO from arginine

(a) RSNO production (ΔRSNO) in control vs. ASA fibroblasts supplemented with L-arginine (1 mM) for 24 hours (*p<0.05). (b) Nitrite production (ΔNitrite) in control vs. ASA fibroblasts in response to listed treatments over 30 minutes (ANOVA, *p<0.05 **p<0.0005). (c) Human ASA subjects (n=3) plasma arginine, citrulline, nitrite and RSNO levels as compared to controls. (*p<0.05 **p<0.005 ***p<0.0005). (d) Dynamic metabolite flux measurements in ASA vs. control subjects. Left panel: Arginine and urea flux in ASA subjects (n=3) vs. controls (n=3). Right panel: Citrulline flux, fractional transfer of the amido-nitrogen from 15N-glutamine to 15N-citrulline, and fractional transfer of guanidine nitrogen from 15N2-arginine to 15N-citrulline (a marker of NO production) in ASA subjects compared to controls, (GLN-Glutamine; CIT- Citrulline, ARG- Arginine) (*p<0.05 **p<0.005 ***p<0.0005 **** p<0.00005). (e) Brachial artery (BA) mean percentage vasodilatation measured using Doppler ultrasound. Left Panel: NOS dependent flow mediated relaxation. The green dashed line represents the lower normal limit for vessel dilation (*p<0.05). Right Panel: NOS independent relaxation stimulated by 0.4 mg of sublingual nitroglycerin. ASA subjects (n=2), controls (n=3). BA - Brachial Artery; NTG - nitroglycerin.

Figure 6. ASL is required to maintain an NO synthetic complex

(a) Immunoprecipitation with antibody to ASS and subsequent immunoblots (IB) with listed antibodies of lung protein lysates from AslNeo/Neo (lanes 1–2) and WT mice (lanes 3–4) (n=2 each). On the right is the densitometric quantification from 3 separate studies performed from a total of 9 WT and 12 AslNeo/Neo mice (*p<0.05). (b,c) Effects of ASL catalytic site mutations on complex formation and NO production. (b) Immunoprecipitation with ASS and NOS3 antibodies of COS7 cells transfected with either WT ASL or R236W ASL, empty plasmid or with GFP and NOS3. Left panel: A western analysis showing expression of the ASL and NOS3. Right panel: Immunoprecipitation with antibody to NOS3 or to ASS followed by immunoblot as listed. The blots are representative of three different experiments. (c) Nitrite production in response to arginine in ASA cells expressing catalytically inactive ASL (*p<0.05). Left panel: Western blot showing the expression of ASL in ASA null cells transduced with ASL R113W vs. control fibroblasts. Right panel: Nitrite levels measured at 15 minutes with and without addition of arginine to the medium (*p<0.05). (d) Model for the NOS complex in arginine channeling. Arginine from de novo cellular synthesis by ASL or via transport from extracellular pools by CAT-1 can be channeled to NOS via ASL.