1Departments of Anesthesiology and Physiology & Biophysics, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2Division of Pediatric Respiratory Disease, University of Virginia School of Medicine, Charlottesville, Virginia 22908
NITRIC OXIDE (NO, nitrogen monoxide) affects a multitude of physiological systems. It can react with metal centers, oxygen, and superoxide in tissues. The report from Zhang and Hogg, one of the current articles in focus (Ref. 13, see p. L467 in this issue), confirms that one cellular target of oxidized form(s) of NO is thiol groups (RSH) (13). Nitrosothiols (RSNO) are formed from peptide or protein RSH (cysteine) residues in the presence of an electron acceptor such as copper (i.e., in ceruloplasmin), iron, or oxygen (5, 11, 13). Recent reports suggest that different cysteine thiols in proteins exhibit different extents of nitrosation (4, 9, 11). Here, Zhang and Hogg confirm previous data (3) that cellular RSNOs are formed under physiological conditions and that this synthesis is increased in association with nitric oxide synthase (NOS) activation. However, the role of RSNO formation in cell signaling remains controversial in part because 1) the mechanisms and kinetics of cellular S-NO bond formation and cleavage in situ are not well defined; and 2) assaying for specific cellular RSNO remains cumbersome, complex, and controversial. For this reason, we propose criteria (Table 1) to help to guide research regarding whether a bioactivity results from the S-nitrosation (or denitrosation) of a specific protein.
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When Zhang and Hogg (13) stopped NO synthesis with a NOS inhibitor, the intracellular RSNO levels declined only slowly, by 50% after 3 h. This result demonstrates that certain protein thiols denitrosate relatively slowly. However, specific protein thiols that may be functionally regulated by nitrosation may well turn over (both nitrosation and denitrosation) rapidly and would not be detected by the methods applied here.
Zhang and Hogg (13) also found that addition of extracellular oxyhemoglobin for 2 h halved the amount of LPS-induced intracellular RSNO protein in cytosolic extracts. Hemoglobin is an efficient NO scavenger that will increase the rate at which NO is lost from the hemoglobin-free side of a membrane (7, 8, 12). It does this by decreasing the probability (to virtually 0) that a molecule introduced to the nonhemoglobin side will, after it diffuses to the hemoglobin side, diffuse back. Therefore, the difference in intracellular RSNO concentration in the presence of extracellular hemoglobin is accounted for by loss of NO molecules that diffused out, but not back (7, 8). It is proposed (Lancaster) that this observation demonstrates that all RSNO formation must be accounted for by NO that diffuses out and back across the cell membrane because this diffusion is more rapid than any NO oxidation process that would form RSNO; thus, rapid diffusion suggests that RSNO formation is not spatially constrained (2) by the location of NOS isoforms, electron acceptors, or target thiols. Indeed, this accounts for data in hepatocytes (2, 10).
On the other hand, it can be argued (Gaston) that RSNO synthesis may be spatially constrained in that 1) it is most likely to occur in membranes and organelles (9), which were not analyzed in the cytosolic extracts prepared by Zhang and Hogg (13); 2) the establishment of a transmembrane pressure/concentration gradient, and consequent increased net transmembrane flow, does not demonstrate that NO must leave and return to react, particularly in the context of rapid intracellular reactions with superoxide, metal-containing proteins, and redox-active amino acid groups; and 3) in any event, spatial constraint of RSNO formation does not necessarily imply spatial confinement of NO. As is the case with all reactions in biology, spatial constraint must not violate the laws of diffusion, but the two principles are not necessarily mutually exclusive: for example, NO diffusing out of the cell can be "spatially constrained" by hemoglobin not to diffuse back. Interestingly, inducible NOS upregulation in the lung, associated with a general increase in nitrotyrosine immunostaining, is associated with localized decreases in RSNO immunostaining, suggesting that RSNO formation is localized and/or metabolically regulated (1). We propose that this differential localization of reactivity might be called "spatial confinement" as opposed to "spatial constraint." Certainly, an important finding of Zhang and Hogg is that NO diffusion affects cellular RSNO concentrations and that RSNO metabolism is best studied in the physiological context in which the cell would find itself in vivo. For example, cellular RSNO metabolism might be expected to be different in a cell exposed to continuous blood flow than in a cell in the center of an abscess.
The cytosolic RSNO in lysates identified by Zhang and Hogg (13) were primarily composed of high-molecular-weight species (>3 kDa). It would have been of interest to know which specific cytosolic proteins were S-nitrosated in the RAW 264.7 cells following LPS exposure. One of us (Gaston, unpublished observations) has found that when assaying protein RSNOs in biological samples, several variables can affect the signal readout; these include preassay protein preparation, position and reactivity of the RSNO bond in the protein, autocapture of NO by metal centers, and other competing reactions of NO. Thus standard curves using S-nitrosoglutathione or S-nitrosated albumin may under- or overestimate the concentration of specific RSNO proteins immunoprecipitated or otherwise isolated from cells or tissues, and quantitative validation by three different, independent assays is ideal at this stage of the science. In this regard, it is of interest that Zhang and Hogg report cytosolic RSNO proteins in greater abundance and having greater stability in RAW 264.7 cells than have previously been reported in other cells (4, 9, 11). Additional work will be required to carefully dissect the specific proteins involved using immunoprecipitation, site-directed mutagenesis, and proteomic techniques (4, 6, 9).
So, three major take-home messages from the work of Zhang and Hogg (13) are that 1) confirming previous studies, protein RSNOs are formed in macrophage cell line cytosolic extracts following LPS stimulation; 2) these protein RSNOs can be quite stable, despite the cytosolic "reducing environment"; and 3) intracellular RSNO metabolism needs to be studied in the physiological context because of the powerful effects of tissue/environmental reactive species, such as hemoglobin, on NO diffusion. It will be of great interest to see 1) if their results are relevant to specific "sequestered" cell compartments; 2) which specific proteins are S-nitrosated in response to LPS stimulation; and 3) what the specific mechanism(s) might be for this S-nitrosation.
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