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1
Chapter
Determinants of Nitric
Oxide Chemistry: Impact of
Cell Signaling processes
Douglas D. thomas1, Wilmarie Flores-Santana2, Christopher h. Switzer2,
David a. Wink2 and Lisa a. ridnour2
1Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy University
of Illinois at Chicago, Illinois
2Radiation Biology Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA
abStraCt
Nitric oxide (NO) is a signaling mediator with diverse as well as opposing biological activi-
ties. The complexity of the NO response reflects the variety of its chemical reactions and
biological properties. Steady-state NO concentration is a key determinant of its biological
outcome as precise cellular responses are differentially regulated by specific NO concentra-
tions. In general, lower NO concentrations promote cell survival and proliferation, while
higher levels favor cell cycle arrest, apoptosis, and/or senescence. Free radical interactions
also influence NO signaling by reducing NO bioavailability. Reactive nitrogen species
generated during these processes also have biological effects by increasing oxidative and
nitrosative stress responses. Moreover, a number of factors influence NO formation and con-
centration, including diffusion, consumption, and substrate availability, which are referred
to as kinetic determinants for molecular target interactions. Kinetic determinants are the
chemical and biochemical parameters that shape cellular responses to NO. Herein we dis-
cuss these chemical interactions and their influence on NO signaling and cellular response.
Keywords: nitric oxide; oxidative; nitrosative stress
INtrODuCtION
Over the last two decades, nitric oxide (NO) has emerged as one of the most diverse and
important ubiquitous biological mediators. It plays a functional role in processes ranging
from neurological function and vascular tonicity to pathogen eradication. NO is unique
in that many of its actions appear to be dichotomous: NO can exhibit distinctly different
Nitric Oxide: Biology and Pathobiology, second edition
Copyright © 2010 by Academic Press. Inc. All rights of reproduction in any form reserved.
SeCtION I
Chemical Biology
and often opposing behaviors under seemingly similar pathological situations. For exam-
ple, murine models have demonstrated that NO synthase (NOS2) knockout mice exhibit
increased incidences of cancers such as leukemia and lymphoma, whereas other studies have
shown that elevated NOS2 protein levels in human breast cancer correlate with poor out-
come. Also, ischemia/reperfusion injury models have demonstrated an improved prognosis
in NOS2 knockouts, while NO donors or nitrite delivered during reperfusion ameliorate this
injury. Paradoxes like these have led to contradictions in the literature. Fortunately, many of
these inconsistencies can be explained by considering experimental details in relation to NO
chemistry.
The physiologic outcome in response to NO is determined by its chemistry. Unlike protein
receptor interactions or small molecules, NO is a diatomic radical that seeks covalent bond
formation(s). Therefore, microenvironmental conditions which determine the biological
outcome of NO exposure are quite different compared to simple structure–function relation-
ships. Toward this end, the chemistry of NO and associated reactive nitrogen species (RNS)
is rich and diverse. There are numerous possible reactive motifs for NO in vivo that are capa-
ble of providing both regulatory and pathologic opportunities. This chemical diversity has
evolved into the daunting task of reconciling the numerous biological effects of NO with its
microenvironment-dependent chemistry.
By defining the chemical biology of NO, we have attempted to provide a description which
in part explains the dichotomous behavior of NO in biological systems. This discussion pro-
vides the framework to explain potential chemical reactions in terms of concentration and
temporal dependence of NO. Biological reactions of NO can be classified into two types as
shown in Fig. 1: reactions mediated through direct effects and those attributed to indirect
effects. The direct effects occur at low NO concentrations and pertain to direct chemical
interactions of NO with its biological targets. These include, for example, reactions with
metal complexes and other radical species. Indirect effects involve RNS derived under con-
ditions of relatively high NO concentration. These include species such as NO and N O
2 2 3
which are derived from the reaction of NO with either superoxide or molecular oxygen. It is
these reactive intermediates, and not NO itself, that go on to target specific motifs.
One might predict potential deleterious effects associated with formation of RNS; however,
these intermediates can mediate important signaling events. For example, thiol oxidation
status dictates the effects of regulatory proteins, including PTPase, PTEN and PP2A, which
FIgure 1
Concentration-dependent direct
and indirect effects of NO. NO Indirect effects
At High Concentrations
> 400 nM
Reactive Nitrogen
Direct effects Species (RNS):
NO, NO derived from
At Low Concentrations reac2tion2 w3ith O and O-.
2 2
< 200 nM
Interaction with:
Metals Complexes
Radical species
Chapter 1
No Concentrations and Signal Transduction
mediate a variety of processes including proliferation and cell survival. Nitrosation of cas-
pases leads to their enzymatic inhibition and subsequent prevention of apoptosis. While
under certain circumstances, apoptosis inhibition may lead to fixed damage and cellular
transformation, the outcome of reactive oxygen species (ROS)/RNS generation is not always
clear. A closer examination of the concentration fluxes of NO has revealed that discrete levels
of NO mediate different signaling pathways. Through the use of well-characterized chemi-
cal NO donors, the concentration-dependent and temporally dependent responses of signal
transduction pathways in response to NO have been elucidated. This information provides
an opportunity to understand the chemical microenvironment at the cellular level. For
example, by comparing cell signaling and steady-state NO profiles of macrophage
co-cultures with those obtained using NO donors, we can begin to estimate NO microenviron-
mental conditions as they relate to specific cellular responses. From this we can understand
how the chemical biology of NO results in a distinct and precise physiologic outcome.
CONtext DepeNDeNCe OF NO reSpONSe
Initially, NO research focused on chemical and biochemical modifications of biological
targets. However as the understanding of NO biology and physiology increased, it became
apparent that the chemistry by which NO elicited specific biological responses was highly
dependent upon its concentration. Upon examination of a variety of proteins known to be
post-translationally modified by NO/RNS, it was revealed that redox chemistry distinguishes
NO response(s). Exposure of MCF7 cells to different NO concentrations for defined periods
of time demonstrated distinct concentration dependence of several signaling proteins
(Fig. 2) (Thomas et al., 2006a,b). Sustained NO levels of 10–30 nM were associated with
cGMP-dependent phosphorylation of ERK in both MCF7 and endothelial cells, while
30–60 nM NO led to Akt phosphorylation (Thomas et al., 2004; Pervin et al., 2007; Prueitt
et al., 2007). Sustained NO concentrations reaching 100 nM resulted in hypoxia inducible
factor (HIF-1) stabilization (Thomas et al., 2004). At concentrations of 400 nM NO, p53
can be both phosphorylated and acetylated. Nitrosation of critical proteins such as PARP
(poly (ADP-ribose) polymerase) and caspase enzymes occurs at 1 M NO and above (sum-
marized in Ridnour et al., 2004). This higher NO level is known to inhibit mitochondrial
respiration (Cleeter et al., 1994; Borutaite and Brown, 2006).
FIgure 2
NO mediates distinct signaling
Nitrosative profiles in a concentration-
HIF-1α P-p53
Stress dependent manner.
P-ERK
Akt-P
10-30 nM
100 nM
400 nM
30-60 nM
0 1 2 3 4 5 6 7 8 9
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
[NO] nM
SeCtION I
Chemical Biology
These and other studies provide insight regarding the dichotomous nature of NO responses
under various microenvironmental conditions and suggest that they are context dependent.
The first level of signaling involves cGMP where low doses of NO (1–30 nM) mediate pro-
liferative as well as protective effects. These levels may be lower in endothelial cells as steady-
state NO fluxes of less than 1 nM can induce cellular proliferation (Isenberg et al., 2005). As
NO levels increase, Akt phosphorylation (Pervin et al., 2003, Pervin et al., 2007, Prueitt et al.,
2007) confers protection against apoptosis through the phosphorylation and inactivation of
Bad and caspase-9 (Chong et al., 2005). At higher NO levels, stabilization of HIF-1 can ini-
tiate proliferative and angiogenic responses and is known to confer protection against tissue
injury (Brahimi-Horn and Pouyssegur, 2007). Levels of NO that are high enough to induce
p53 phosphorylation result in cytostasis and even apoptosis (Hussain et al., 2003). These
observations suggest that low concentrations of NO tend to favor pro-growth and anti-apop-
totic responses while higher levels favor pathways leading to cell cycle arrest or apoptosis.
In addition to concentration, the duration of NO exposure is equally important. While
certain proteins respond immediately to NO, others require hours or even days for activa-
tion. For example, HIF-1 responds rapidly to NO and requires sustained threshold levels
(Fig. 2). When NO levels fall below the minimum concentration threshold necessary to
regulate HIF-1 the protein disappears. This suggests that under normoxic conditions, HIF-
1 is a real-time dosimeter of NO concentration, at least in cells grown in culture (Thomas
et al., 2004; Thomas et al., 2006a; Thomas et al., 2006b). In contrast, the phosphorylation of
p53 in response to NO (400 nM) takes several hours but is sustained even after NO levels
have dissipated. Therefore, we have proteins that are ‘immediate responders’ and those that
are ‘delayed responders’, which can be further classified into proteins that are ‘transient’ or
‘sustained’ responders. HIF-1 falls into the category of an immediate-transient responder,
whereas p53 is a delayed-sustained responder. Interestingly, while phosphorylation of ERK
via cGMP qualifies as an immediate responder, it is also sustained after the loss of NO.
While these classifications are purely functional and somewhat subjective, they are useful
when deciphering mechanisms of protein regulation by NO.
As the biological responses of NO are multifaceted and appear contradictory at times, NO
concentration dependence may help to clarify such discrepancies and point to a less com-
plicated model. Many of the observed differential cellular responses to NO may be simply a
function of its concentration and duration of exposure, so that distinct biological responses
result from specific incremental NO levels. NO is a highly diffusible molecule whose biolog-
ical concentration is determined partially by its distance from the point of synthesis as well
as the cellular redox environment as discussed below. This suggests that the rate of NO
production is a critical determinant of cellular responses. Most of the current data have
focused on whether NO can regulate specific signal transduction pathways, cause chemical
modifications, or inhibit specific enzymes. Until now, little was known regarding NO
concentration dependence and cellular response mechanisms.
CeLLuLar mILIeu aND NO reDOx ChemIStry
Superoxide (O) and the subsequent formation of peroxide (hydro- or alkyl-) are involved
2
in many disease processes (Halliwell and Gutteridge, 1999). Superoxide is generated under
a variety of normal and pathological conditions and induces many signal transduction path-
ways (Forman et al., 2002). It can activate metal complexes and promote damage through
redox cycling and Fenton chemistry. Similarly, peroxide reacts with metals and initiates
phosphorylation cascades. Nitric oxide has been shown to have antioxidant properties by its
diffusion-controlled reaction with O (Wink et al., 2001). This reaction prevents the redox
2
chemistry of O and inhibits H O formation. An interesting relationship involves the effect
2 2 2
of O and H O on NO signaling. While NO inhibits the effects of O , it appears that O
2 2 2 2 2
also ablates NO signaling (Brune, 2005). For example, the generation of O during NO
2
Chapter 1
No Concentrations and Signal Transduction
exposure regulates its concentration and thus affects the response of downstream NO targets
in a concentration-dependent manner. Since certain regulatory proteins respond to distinct
threshold concentrations of NO, the consequence of O being present is a diminution in
2
NO concentration which effectively converts a cytostatic phenotype to one of pro-growth. For
instance, the presence of O during exposure of MCF7 cells to high levels of NO (500 nM)
2
attenuated NO-mediated p53 phosphorylation but maintained HIF-1 stabilization (Thomas
et al., 2006a,b). Conversely, if O fluxes are held constant and NO levels are increased,
2
phosphorylation of both p53 and HIF-1 signaling is attenuated. Since NO is freely dif-
fusible, intercellular or extracellular generation of superoxide had the same effect. These
results demonstrate the mutual ability of ROS and NO to co-regulate signaling mechanisms.
Consequently, superoxide dismutase (SOD) can reverse these responses by increasing the
bioavailability of NO when superoxide is present. Examination of soluble guanylate cyclase
(sGC) activation and cGMP production by NO demonstrated an increase in the EC for nitric
50
oxide when measured in the presence of O . SOD, via O removal, was able to restore
2 2
the responses to NO by shifting the EC for sGC to considerably lower NO concentrations
50
(Thomas et al., 2006a,b). These results suggest one mechanism by which tumor cell NADPH
oxidase (NOX) and SOD affect NO signaling.
Even though the interactions of ROS with NO results in RNS that can have their own effects
on biological pathways, the dominant outcome of these reactions is the scavenging of NO
by its innocuous conversion to nitrite/nitrate. Although the initial chemical species formed
from NO reacting with O is peroxynitrite (ONOO), the presence of CO catalyzes its
2 2
rapid conversion to other species. When the flux of NO exceeds that of O , NO will react
2
with ONOO, resulting in the predominant formation of NO and N O . Conversely, when
2 2 3
O is in excess, ONOO-mediated oxidation and nitration are quenched, suggesting that
2
direct ONOO chemistry has a limited role. Moreover, it has been shown that NO gener-
2
ated from this reaction can oxidize, nitrate, and nitrosate proteins via N O (Espey et al.,
2 3
2002a,b) indicating that it may be involved in cell signaling. Drapiers and coworkers
showed that NO and N O could induce heme oxygenase (HO)-1 and interleukin (IL)-8
2 2 3
in monocytes (Turpaev et al., 2004), implicating a role for RNS in both anti-inflammatory
and pro-growth pathways. Other studies have shown activation of transforming growth
factor (TGF)- by RNS (Vodovotz et al., 1999) thereby down-regulating Th1 type inflamma-
tion. Therefore, despite the potential for deleterious effects of RNS, biological signaling has
adapted to these reactive intermediates to utilize a feedback mechanism which suppresses
the inflammatory response to promote wound healing and tissue restoration. RNS-mediated
cellular responses indicate the presence of biological ‘alarm systems’ that are important in
understanding the in vivo NO response.
CONCeNtratION raNge OF eNDOgeNOuSLy geNerateD NO
Although NO elicits many unique cellular responses in vitro, the question remains, are these
NO concentrations and conditions actually achievable in vivo? Several studies have employed
different methods to examine specific NO concentrations under various biological condi-
tions in vivo (Tarpey et al., 2004; Zhang and Hogg, 2004). One approach is to compare the
cellular response(s) to NO generated from known concentrations of NO donor compounds
to those responses associated with the induction of NOS. Experience with NO donor com-
pounds has shown that individual targets demonstrate unique concentration sensitivity to
NO. By measuring biological effects of NO and correlating them with measured NO concen-
trations in vitro, we can approximate the in vivo NO concentrations necessary to elicit specific
cellular responses. Using this ‘molecular signature’ approach for various concentrations of
NO, the redox microenvironment can be determined as it relates to specific biological condi-
tions. For example, MCF7 cells co-cultured with activated NO-producing macrophages result
in increased phosphorylation of Akt and p53, as well as stabilization of HIF-1 (Hofseth
et al., 2003; Thomas et al., 2004; Prueitt et al., 2007). As the macrophage number is
SeCtION I
Chemical Biology
increased, a proportional increase in the concentration of NO can be measured in solution
(Thomas et al., 2004; Prueitt et al., 2007). At each increase in the ratio of macrophages to
MCF7 cells there was a concomitant increase in pAkt (Prueitt et al., 2007), HIF-1 stabiliza-
tion, and p53 phosphorylation which correlated to increasing NO concentrations (Thomas
et al., 2004). This indicates a dose–response effect of endogenously produced NO on target
proteins. Phosphorylation of p53 required at least 400 nM steady-state NO when generated
by the donor Sper/NO, while this threshold dropped to 160 nM during MCF7/macrophage
co-culture experiments (Thomas et al., 2004). This discrepancy can be rectified by consider-
ing that NO generated from donors is uniformly distributed throughout the media, whereas
the enzymatic production of NO from macrophages generates a concentration gradient
emanating from the point of origin. Due to the close proximity of the macrophages to the
MCF7 cells in this example, the actual local concentration of NO is substantially greater than
160 nM and is at least as much as 400 nM (Hofseth et al., 2003; Thomas et al., 2004), which
is the minimum amount of NO necessary to cause p53 phosphorylation.
Macrophages are not only an important part of the immune system but can also be a major
source of NO production. They perform a wide variety of functions from fighting bacteria
and suppressing tumor growth to coordinating the resolution of tissue repair and restora-
tion processes. Macrophages can generate a number of different levels of NO that mediate
different functions (Espey et al., 2000). For instance, eNOS (endothelial NOS) stimulation
of guanylate cyclase in non-activated macrophages is required to fully induce iNOS (induc-
ible NOS) expression in LPS-activated macrophages (Connelly et al., 2003). Interestingly,
the amount of NO produced from activated macrophages is dependent upon the manner by
which they are stimulated. When cultured macrophages are pretreated with interferon (IFN)-
followed by activation with TNF- or IL-1-, the amount of NO measured in solution is
approximately 10 times less than that of IFN- LPS (lipopolysaccharide)-stimulated cells
(polyinosinic-polycytidylic (PIPC) or Listeria) even though there is only a modest difference
in NOS activity (Espey et al., 2000; Ogawa et al., 2001). In addition, the amount of NO-
mediated nitrosation from TNF- or IL-1- stimulation was 30 times less than those stimu-
lated with LPS or PIPC (Espey et al., 2000). This suggests that NO generated from cytokine
stimulation leads to considerably lower NO concentrations in the surrounding environment
as compared to agents that activate NOS through the Toll-like receptors. These examples
demonstrate that the range of NO concentrations generated from macrophages is partially
dependent on the activating agent(s).
Matrix metalloproteinases (MMPs) are important mediators of inflammation, angiogenesis,
wound response, and cancer (Parks, 1999; Chang and Werb, 2001; Sternlicht and Werb,
2001; Coussens and Werb, 2002). MMPs are tightly regulated, primarily at the levels of tran-
scription and post-translation, and are targeted by ROS/RNS at both of these levels (Nelson
and Melendez, 2004; Okamoto et al., 2004; Siwik and Colucci, 2004). While contradictory
results have been reported with respect to redox regulation of MMPs, the ability of ROS and
RNS to biphasically regulate MMP activity has been reported and suggests a critical role of
these species in the spatial and temporal regulation of MMPs during matrix remodeling
(Peppin and Weiss, 1986; Fu et al., 2001; Ridnour et al., 2007). Toward this end, the biphasic
and dose-dependent regulation of matrilysin (MMP-7) by HOCl, initially demonstrated by
Peppin and Weiss (Peppin and Weiss, 1986) involved oxidation of the cysteine switch at low
HOCl concentrations that mediated activation of the zymogen (Fu et al., 2001). High HOCl
concentrations inactivated the enzyme by chlorination oxidation of adjacent tryptophan-gly-
cine residues within the catalytic domain. This led to structural modifications that impeded
substrate binding at the active site cleft (Fu et al., 2003, 2004). The physiologic relevance of
oxidant-mediated MMP inactivation was later demonstrated using gp91phox knockouts in a
murine model of emphysema (Kassim et al., 2005). Macrophage-derived NO has also been
shown to biphasically modulate the activity of MMPs. A recent report demonstrated both
biological and chemical mechanisms of MMP activation by macrophage-derived
Chapter 1
No Concentrations and Signal Transduction
NO (Ridnour et al., 2007). The activity of MMPs (MMP-9, -1 and -13) secreted from mac-
rophages increased in response to low steady-state NO levels (50 nM). This occurred via
cGMP-mediated suppression of the endogenous tissue inhibitor of metalloproteinase-1
(TIMP-1) (Ridnour et al., 2007). Exposure of purified latent MMP-9 to exogenous NO dem-
onstrated a concentration-dependent activation (500 nM) and inactivation (1 M) of the
enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentra-
tions similar to that of activated macrophages. Interestingly, TIMP-1 inactivation by HOCl
is mediated by oxidation of an N-terminal cysteine residue that is critical for binding at the
MMP active site. This suggests multiple mechanisms for oxidant regulation of MMPs during
matrix remodeling (Wang et al., 2007). In an epithelial wound model, low NO levels simi-
larly increased MMP-9 mRNA, which translated to enhanced enzymatic activity (Bove
et al., 2007). Together, these results suggest that NO regulation of MMP-9 secreted from
macrophages may occur chemically by RNS-mediated protein modification or biologically
through cGMP-dependent modulation of the MMP-9/TIMP-1 balance.
One of the first observations establishing that macrophages and hepatocytes are a source
of NO was the formation of nitrosamines by amine nitrosation (Marletta, 1988; Liu and
Hotchkiss, 1995). This provided evidence that nitrosative stress can be locally generated
from iNOS. Other studies showed that endogenous NO and RNS inhibited dopamine -
hydroxylase. However, NO generated from macrophages co-cultured with neuroblastoma
cells could only be mimicked by M NO donor concentrations that generated M NO
(Zhou et al., 2000). When macrophages were co-cultured with A549 cells, latent TGF- was
converted to active TGF- (Vodovotz et al., 1999). Again, this could only be recapitulated
with NO donors that gave M fluxes of NO. Yet another example is the cytostatic effect of
NO on Listeria (Ogawa et al., 2001). This pathogen propagates in the macrophage. When
the thiol-dependent protease becomes nitrosated by NO generated from iNOS, the organ-
ism is unable to escape the macrophage. Similarly, this NO microenvironmental effect could
only be replicated by mM amounts of an NO donor that produced M steady-state NO
concentrations for a prolonged period of time. In a study of astrocytes co-cultured with
human microglial cells infected with HIV, iNOS activity was essential to inhibit the reverse
transcriptase activity (Hori et al., 1999). Again, to mimic this, mM amounts of an NO donor
were required.
While the indirect effects of these high steady-state NO levels can be achieved in culture,
the important question remains as to whether or not they occur in vivo. Several studies have
indicated that in murine and human models these upper level fluxes of NO are indeed
achieved in tissue. Inhibition of cytochrome P450 and cyclooxygenase (COX)-2 has pro-
vided evidence that high levels of NO can be achieved in the vicinity of inflamed liver cells
(Stadler et al., 1993, 1994; Snyder et al., 1996). NO-mediated inhibition of P450 may have
important consequences under inflammatory conditions (Yaghi et al., 2004; Tunctan et al.,
2006). During sepsis or endotoxemia, P450 inhibition has been found to correlate with
NOS expression (Muller et al., 1996; Takemura et al., 1999; Carcillo et al., 2003; Lee and Lee,
2005). Moreover, the effective inhibition of P450 activity requires 100 nM to M levels of
NO. While oxygen tension in vivo may change this value, these results nevertheless indicate
that high levels of NO can be achieved under inflammatory conditions.
Clinical evidence suggests that high M NO levels are achieved in humans during inflam-
matory pathologies. Immunohistochemical analyses have demonstrated co-localization of
iNOS and P53 phosphorylated at ser-15 in patients with ulcerative colitis. Also, a correlation
between iNOS expression and HIF-1 stabilization was reported in human colorectal cancer,
indicating the generation of 100 nM NO (Yu et al., 2006). These reports suggest that indi-
rect effects of NO occur under inflammatory conditions.
Nitrosamines, including nitrosoproline, are generated in vivo under septic conditions
(Wishnok et al., 1993; Mirvish, 1995). Nitrosamine formation can be achieved by two distinct
SeCtION I
Chemical Biology
mechanisms: nitrosation and oxidative nitrosylation. Oxidative nitrosylation involves sub-
strate oxidation leading to radical formation, which then couples with NO to form the nitro-
sated product. Oxidative nitrosylation occurs under very limited conditions with amines that
have low oxidation potentials. Although this is chemically plausible, it remains to be demon-
strated under biological conditions. In contrast, nitrosation can occur through direct addition
of a nitrosonium equivalent (NO) from RNS such as N O . Proline and other secondary
2 3
amines have high oxidation potentials (1.5 V) and therefore undergo exclusively nitrosative
chemistry. Studies have shown increased nitrosated thiols (RSNO) in human lung patholo-
gies indicating that nitrosative species are generated in vivo (Gaston et al., 1993).
Another indirect indicator of RNS formation is the presence of 3-nitrotyrosine (3-NT).
Immunohistochemical staining for this species has been used as a footprint for NO and RNS
in tissue. While peroxynitrite had long been assumed to be the sole source of 3-NT, further
investigation revealed that this species is likely not responsible for all 3-NT observed in vivo
(Pfeiffer et al., 2001a,b). Oxidation of nitrite by one electron to form NO through Fenton
2
type or peroxidase chemistry provides an alternate, more physiologically relevant mechanism
for the generation of 3-NT (Eiserich et al., 1998; Thomas et al., 2002). NO is unique in that
2
if generated in the absence of NO it can migrate through cells (Espey et al., 2001). This radi-
cal is thiophilic and may initiate signal transduction pathways such as antioxidant response
element (ARE) activation (Ford et al., 2002). Unlike O , which is an anion, and the OH
2
radical, which reacts at its point of generation, NO can readily diffuse to adjacent cells. This
2
provides a mechanism for NO -mediated signal transduction in vivo. Thus, 3-NT may repre-
2
sent the formation of NO from nitrite rather than high levels of NO gas. Therefore, ample
2
evidence exists supporting a role for the indirect effects of NO in vivo, which are mediated by
high NO levels during inflammation.
Nitric oxide mediates an array of responses in the nervous system, such as dopamine release
10
in the striatum (Hanbauer et al., 1994). Interestingly, the replication of this response in syn-
aptosomes and isolated cells required an NO flux of nearly 1 M in concentration, such as
that provided by a rapid-release NO donor (DEA/NO or Sper/NO). This suggests that local
mediation of dopamine release by NO may require rapid threshold bursts of higher levels
of NO (200 nM) generated near the postsynaptic neurons. Importantly, the high threshold
requirements for this response indicate that lower levels of NO derived from other sources
will not randomly activate this response. Thus, within the synaptosomes microenvironment,
nNOS can likely achieve NO levels 100 times higher than required to stimulate vasorelaxation
of vascular smooth muscle cells, which suggests that higher NO concentrations can be used to
mediate specific physiological responses in a very confined area over short periods of time.
Buerk and coworkers have used microelectrodes to successfully measure similar high NO
levels (300 nM) following N-methyl-D-aspartate (NMDA) stimulation of the cerebral cortex
(Thom et al., 2004). Moreover, during NMDA stimulation, the translocation of NOS to Ca2
channels and subsequent RNS generation may be an important event under inflammatory
conditions by protecting neurons from Ca2 overload due to increased release of glutamate.
Toward this end, N O -mediated nitrosation of thiols and subsequent closure of the NMDA
2 3
channel have been shown, suggesting that high NO levels may mediate this response in iso-
lated regions of the cell (Lipton et al., 1993; Gbadegesin et al., 1999). Another interesting
aspect of NO regulation of the NMDA channel under oxygen/glucose-deprivation conditions
is that low doses of NO mediate increased opening of the channel (Gbadegesin et al., 1999).
In summary, it is plausible that direct effects of NO modulate an increase in channel func-
tion, while thiol nitrosation mediated by indirect effects of NO closes the channel and pro-
tects against glutamate toxicity.
The examples cited above demonstrate an upper range of NO concentrations in vivo, which can
be mimicked by mM concentrations of NO donors. However, the question remains: What are
the lower concentration limits of NO that mediate biological effects? The relaxation of vascular
