Our findings support a requirement for ASL not only to synthesize intracellular arginine, but also to utilize extracellular arginine for NOS-dependent NO synthesis. We demonstrate an intracytosolic complex of proteins important for nitric oxide synthesis to explain the structural basis for metabolite channeling that reconciles the phenotypes observed in human and mouse models of ASL deficiency. While likely not the only explanation, the data support that decreased ASL levels leads to loss of NOS complex formation that is associated with NO deficiency at the whole organism, tissue and cellular levels in both humans and mice.
This conclusion is based on a combination of mouse and human studies that were prompted by clinical observations in ASA patients. First, the natural history of ASL-deficient humans with argininosuccinic aciduria suggested the presence of organ dysfunction and complications that were independent of hyperammonemia caused by hepatic urea cycle deficiency. This led us to hypothesize alternative mechanisms of injury including deficiency of NO secondary to loss of citrulline recycling or endogenous arginine production. However, this could not alone explain the phenotypic complexity because ASL patients are replete with extracellular arginine due to pharmacological supplementation.
This apparent conundrum was evident in the hypomorphic mouse model of Asl deficiency, where we observed histological evidence of multi-organ dysfunction that correlated with biochemical evidence of systemic NO deficiency. This was further supported by the finding of decreased markers of NO production (plasma RSNO and nitrite) in ASA patients. Importantly, ASA patients, ASA fibroblasts, and primary endothelial cells made deficient of Asl by siRNA knockdown, were also unable to efficiently generate NO after extracellular arginine supplementation. In humans, this was demonstrated via dynamic measurement of arginine to citrulline flux; while in cells, it was demonstrated via measurement of nitrite and/or cGMP. This NO deficiency was found to be tissue autonomous as preconstricted aortic rings from mutant mice relaxed in response to NO donors but not arginine, while ASA subjects exhibited abnormal flow-mediated vascular relaxation that was restored by nitroglycerin. On the whole organism level, provision of an NOS independent source of NO in the form of nitrite therapy significantly prolonged survival of hypomorphic mice while also restoring tissue nitrosylation and normalizing blood pressure.
The NO deficiency was caused in part by the inability of patients and cells to efficiently generate intracellular arginine or to utilize extracellular arginine for NO production in the face of ASL deficiency. This was in spite of adequate expression of all other protein components necessary for NO production including the arginine transporter CAT-1, ASS, HSP90, and NOS. This observation correlated with the existence of a NOS complex that depends on the structural, but not enzymatic function of ASL. Loss of ASL was associated with decreased abundance of this complex, decreased utilization of arginine for NO production, and functional consequences of NO deficiency at the organ and cellular levels.
This structural requirement was supported by the ability of specific mutations in the ASL catalytic domain to participate in the NOS complex while the complete absence of ASL prevented efficient complex formation. Together, these studies distinguish two essential roles for ASL: the recycling of citrulline in the cell for cell autonomous arginine synthesis, and the maintenance of a NOS complex that is required for efficient NO production from extracellular sources of arginine. The former depends on the catalytic function of ASL, while the latter requires its structural integrity. Interestingly, this is consistent with the distinct evolutionary roles of ASL in other species33
. Since in cell, mouse, and patient models, the NO deficiency are evidenced in the face of excess arginine, it is likely that the primary dysfunction is at the level of the NO metabolon where insufficient channeling of arginine due to loss of the ASL-NOS complex leads to secondary decreased NO production. An important question for future study is whether the loss of arginine channeling leads to NOS uncoupling and consequent increase in free radical stress.
Clinically, these data suggest that the vascular dysfunction and intellectual delay seen in ASA patients may be partly due to NO insufficiency, and hence, treatment with a NOS-independent source of NO, e.g., sodium nitrite, or NO donors, would be beneficial in the long term. Moreover, the clinical variability seen in ASA patients may depend on the differential effects of specific mutations on catalytic vs. structural functions.
These data also have broader implications for NO biology and disease. Mechanistically, they support intracellular compartmentalization as an explanation for the “arginine paradox”, i.e., the increased production of NO with the addition of extracellular arginine despite apparently saturating intracellular arginine levels. Moreover, they suggest an explanation as to why the arginine paradox is not observed with ASL deficiency. As such, ASL may serve as the linchpin in NO production. Hence, inhibition of ASL in a cell-specific fashion may be an effective way to probe NO function in vivo, independent of potential NOS redundancy. Similarly, it may serve as a novel target for manipulating NO production in a cell autonomous fashion in human disease processes.