Heme nitrosylation (16
) and S-nitrosation (14
) represent two protein modifications that account for much of the signaling action that NO exerts in cells. Both types of post-translational modifications are effected through NO or NO-derived substances (20
), including nitrite (3
). In all cases, the sources of these substances are associated with endogenous NO synthesis and metabolism (2
) or dietary NOx intake (5
). Consequently, our finding that protein-bound NO modifications persist in the absence of active NO synthesis and dietary NOx intake (e.g., in eNOS−/−
animals on a low-NOx diet and under the acute action of the NOS-inhibitor, L-NIO, or in Wistar rats on low-NOx diet with chronic administration of L-NMMA) is surprising since it suggests the existence of an unrecognized, and significant, source that contributes to the formation of NO signaling products. Our results may also explain why nitrosyl hemoglobin levels only dropped by half following dietary NOx restriction and complete NOS inhibition in another study (5
). Although our results cannot pinpoint the precise nature of this new source of NO products, our results hint at the intermediacy of nitrite in the process, as we shall discuss below.
Our finding that protein-bound NO levels remain essentially unchanged upon deletion of each NOS isoform (, lower panels) likely reflects the action of a compensatory mechanism that maintains basal levels of NO metabolites in tissues. One obvious compensatory mechanism that would keep tissue NO levels constant is upregulation of remaining NOSs in an attempt to restore basal levels. If upregulation of alternative NOS isoforms alone were to take place, then there would be no physical reason why nitrite levels should change, according to the chemical network depicted in . Yet, our results shown in clearly indicate that nitrite is consistently depleted, regardless of the nature of the specific NOS isoform deleted. These consistent nitrite reductions suggest that increased utilization of nitrite may be a significant component of the regulatory mechanism associated with deficient NOS expression/NOS deletion.
If inhibition of any one NOS isoform leads to increased nitrite consumption, then it must also be accompanied by increased production of nitrite elsewhere if tissues are to maintain a nonvanishing level of this anion. According to the nitrate-nitrite-NO pathway that has captured much attention recently (17
), such increased nitrite production could originate from the reduction of nitrate. However, the converted nitrate would also have to be replenished by some other source to sustain the pathway. For animals on a normal diet, such as those featured in , it could be argued that the pathway may be sustained by NOx extracted from the diet. Yet our results for NOS-inhibited animals on a low-NOx diet (–) reveal no evidence that effective (>98%) dietary restriction of NOx intake leads to substantial suppression of tissue nitrite or nitrate levels. This would argue that any increased production of nitrite from nitrate would ultimately have to be derived from some source not considered in our scheme depicted in .
The notion of NOS-independent NO production in mammalian tissues is not new (37
). However, the effects we here observe using NOS knockout animals are larger than usually assumed. Since rodents synthesize ascorbic acid, and ascorbate has been shown to promote NO generation from nitrite reduction, either by direct chemical reaction (28
) or by xanthine oxidase-catalyzed reduction in tissues (18
), adaptive changes in ascorbate levels may have taken place to attenuate the drop of NOS-dependent NO production. This possibility was investigated in rats subjected to chronic NOS inhibition rather than by comparing wild-type with NOS−/−
mice. In most compartments, changes in ascorbate content in response to restriction of NOx intake and chronic NOS inhibition were moderate. Only in the aorta, ascorbate increased significantly while glutathione levels dropped, and even these changes are not accompanied by nitrite depletion. Thus, the data does not support the notion that tissue nitrite reduction is enhanced due to increased ascorbate content and/or recycling. Moreover, administration of the xanthine oxidase inhibitor allopurinol did not alter nitrite levels significantly (not shown). Taken together, these results suggest that neither ascorbate nor xanthine oxidase make a major contribution to total NO product formation in vivo
Whether nitrite plays a central role in sustaining NO signaling in the absence of NOS activity, an inescapable conclusion from our results is that NO-related substances continue to be synthesized in mice even after dietary NOx is highly reduced and NOS is inhibited. Our study in mice is limited inasmuch as we did not extend the period of NOS inhibition beyond 3 hours (in order to avoid stress as a possible confounder). Nevertheless, this period should have been more than enough to see substantial changes in nitrite and protein-bound NO levels as it corresponds to twice the plasma half-life of nitrate in mice (34
), the NO metabolite with the longest half-life in vivo
and the largest pool of NO products in the body. In the absence of other sources of intake or production, whole-body nitrate levels should have dropped by 75% within that time if NOS was completely inhibited. Yet, such level of reduction is not observed. Considering that reversible enzyme inhibitors were used, one could argue that NOS inhibition may have been incomplete. However, our more chronic NOS inhibitor studies in rats yielded similar results. Had the degree of enzyme inhibition in the acute studies in mice and in the chronic studies in rats not both been close to maximal, such similarity in outcome would have been highly unlikely.
An intriguing additional feature emerging from the rat studies was the paradoxical upregulation of NO products during chronic combined dietary NOx restriction and pharmacological NOS inhibition in aortic tissue. The observed increase of NO products in the aorta following NOS inhibition may be secondary to enhanced NOx uptake from the circulation, indicative of an enhanced need for such products in the vasculature when NO production from L-arginine is compromised.
However surprising these findings may be, they can only be interpreted as evidence that a source other than those depicted in may be sustaining the nitros(yl)ation/NOx cycle at basal levels when NOS is inhibited. This source, although unidentified at this time, may be a key to understanding why even triple NOS knockout mice remain viable.
At this stage, we can only speculate about the origin of this elusive source of NO products. Future studies under specific pathogen-free conditions may provide insight as to whether it originates from microbial or mammalian activities. Further investigations, requiring the use of tracer techniques such as 15N-labeling in combination with isotope-specific analytical methods, may reveal that the unknown pool of NO metabolites is derived from the oxidation of ammonia, metabolism of hydroxylamine, uptake/metabolism of a nitrogen species in air, or perhaps “recycling” of urea. Other possibilities may include reductive biotransformation of nitrated species. Alternatively, it may be a by-product of protein catabolism/turnover and derived from the α-amino group of amino acids.
Finally, we wish to point out an interesting observation that may bear on the role of nitrite in NOx-restricted, NOS-deleted animals. As stated earlier, these animals exhibit significantly reduced levels of whole-body nitrite compared to those of WT animals. However, when one examines the concentration changes relative to WT for each organ, the level of reduction varies widely from one organ to another (). In fact, the level of reduction appears to vary systematically with tissue mitochondrial inner surface area (a proxy for maximal oxidative phosphorylative capacity of that tissue; data taken from 15) as suggested in that figure. This observation confirms and extends earlier in vitro
) related to the nitrite reductase activity of the same tissues to the in vivo
situation. This link suggests that the nitrite depletion we observe in tissues may be related to mitochondrial activity and perhaps it holds a key to unlocking the mystery of how basal NO metabolite concentrations (and, presumably, related downstream signaling) is sustained in the absence of NOS and dietary NOx pathways.
FIG. 6. Fractional change in steady-state concentrations of nitrite in organs from eNOS−/− mice maintained on a low-NOx diet, with and without additional NOS inhibition. All values are expressed relative to those found in the same organs of wild-type (more ...)