l-Arginine and Nitric Oxide: An Inseparable Couple?
To the Editor:
Yang and Loscalzo1 recently showed l-arginine–reversible induction of tissue factor (TF, measured as mRNA, protein expression, and activity) in microvascular endothelial cells exposed to endotoxin and interleukin (IL)-1β. The authors conclude that “enhanced production of endothelium-derived nitric oxide (NO) reduces endotoxin- and cytokine-induced expression of TF and, thereby, the prothrombotic phenotype of the endothelial cell.” However, in my opinion, the role of NO, as expressed in the data presented by the authors, is somewhat perplexing.
The authors base their reasoning on the fact that 1 mmol/L l-arginine had inhibiting effects on endotoxin/IL-1β–induced TF activation, whereas the inactive stereoisomer d-arginine and the NO synthase (NOS) antagonist Nω-nitro-l-arginine methyl ester (L-NAME) had no effect. Under those experimental conditions, l-arginine was unlikely to be a rate-limiting factor for NOS activity for the following 3 reasons: (1) l-arginine is normally present in the culture medium, and its concentration ranges from 0.3 mmol/L (Medium 199) to 1 mmol/L (RPMI 1640); (2) the Km value (Michaelis-Menten constant) of NOS for its substrate l-arginine is very low (ie 5 to 10 μmol/L); and (3) no induction of inducible NOS (iNOS), the high-output isoform of the enzyme, or upregulation of endothelial NOS (eNOS), the constitutively expressed isoform of the enzyme, occurred. Nevertheless, a 7.2-fold increase in nitrite/nitrate concentration was measured after 24 hours in the conditioned medium of cells that received 1 mmol/L l-arginine supplementation with respect to control cells, suggesting increased production of NO. It is unclear whether a 45-minute pretreatment of endothelial cells with 1 mmol/L l-arginine followed by a 24-hour treatment with endotoxin/IL-1β resulted in increased NO production and, hence, an accumulation of nitrite/nitrate in the conditioned medium. Moreover, it is unknown whether the observed increase in NO end products was enantiomer-specific and reversible by L-NAME.
L-NAME had no effect on endotoxin/IL-1β–induced TF expression, suggesting basal NO was not involved in TF modulation. Unfortunately, the key experimental group that would have evidenced a NO-mediated mechanism of action for l-arginine (ie, endotoxin/IL-1β plus l-arginine and L-NAME) is missing.
In light of these considerations, a non-NO–mediated role for l-arginine in modulating TF expression may also be hypothesised.
- Copyright © 2001 by American Heart Association
Yang Y, Loscalzo J. Regulation of tissue factor expression in human microvascular endothelial cells by nitric oxide. Circulation.
Dr Bachetti suggests that our data are not inconsistent with an NO-independent effect of l-arginine on tissue factor expression in microvascular endothelial cells. We do not disagree with this interpretation, especially because (as she stated) l-arginine is not likely to be limiting as a source of NO, a point one of us (J.L.) made in a recent Circulation editorial.R1 There are, however, alternate mechanisms by which l-arginine can be limiting, such as in the presence of asymmetric dimethylarginine. We think that the most compelling evidence presented in our article—the stereospecific effects of l-arginine compared with d-arginine, the increase in nitrogen oxides in conditioned media after the incubation of cells with l-arginine, and the similar inhibitory effects observed with an NO-donor (S-nitrosoglutathione)—are best interpreted in toto as consistent with a direct inhibitory effect of endogenous NO on tissue factor expression. Alternative explanations for the effect of l-arginine cannot, of course, be excluded with complete certainty.