Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production
Introduction
Nitric oxide (NO)1 is a critical signaling molecule involved in control of vasomotor tone, vascular homeostasis; neuronal and immunological function [1], [2], [3], [4]. Endogenous NO is produced through the conversion of l-arginine to l-citrulline by NO synthase (NOS) [5], [6], [7]. There are three major isoforms of NOS. The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed and require Ca2+ and calmodulin for activation, whereas the inducible isozyme (iNOS) is largely Ca2+ independent [5]. The expression of iNOS is induced in a wide variety of tissues in response to endotoxin, endogenous mediators of inflammation, and other stimuli such as hypoxia [8]. Relative to the constitutive isoforms, iNOS has ∼5-fold higher NO production.
The active forms of all NOS isozymes are homodimeric. Each monomer of the homodimer is associated with calmodulin (CaM) and contains the bound cofactors BH4, FAD, FMN, and iron protoporphyrin IX (heme) [9]. Each monomer consists of the heme-binding oxygenase domain that also contains the BH4 and l-arginine binding sites, and the reductase domain that contains the NADPH-binding site, FAD and FMN. When CaM is bound to the dimeric NOS, electrons flow from the reductase of one monomer to the oxygenase domain of the other monomer, which produces an activated oxygen species at the heme, leading to substrate monooxygenation. The production of NO from l-arginine by NOS occurs via two sequential monooxygenation events, consuming 1.5 equiv. of NADPH for every NO produced [10].
All three NOS isoforms can generate , depending on substrate and cofactor availability [11], [12], [13], [14], [15]. When NOS is not saturated with the cofactor BH4, each NOS isoform has been shown to catalyze the reduction of oxygen to [14], [15], [16], [17], [18], [19]. In the postischemic heart, BH4 depletion triggers endothelial dysfunction with loss of NO but gain of superoxide production [16]. In l-arginine-depleted macrophages, iNOS generates both and NO leading to peroxynitrite-mediated cell injury [13]. Moreover, iNOS has been implicated in many diseases associated with inflammation [20].
Oxidative stress occurs at low levels normally, but it is greatly enhanced in a variety of diseases associated with inflammation. Reactive oxygen species (ROS) that are commonly formed include , OH, and H2O2. The reactive nitrogen species (RNS) ONOO− is formed when combines with NO. Under normal physiological conditions these species are detoxified by several mechanisms, however, when the ROS and/or RNS are overproduced as occurs in many diseases involving chronic inflammation, these reactive species can cause oxidative damage to cellular proteins, membranes and DNA [21], [22], [23], [24]. The induction of both iNOS and ROS during inflammation is well established [25], [26], however little is known regarding how cellular oxidants affect iNOS function.
In order to characterize the effects of specific reactive oxygen or nitrogen species on iNOS function, the purified enzyme was pre-exposed to known amounts of , OH, H2O2, and ONOO−, and the dose-dependent effects of each oxidant on NO and generation rates were quantified. We observe that , OH, H2O2, and ONOO− all trigger a dose-dependent decrease in NO production. In marked contrast, each of the three ROS species led to increased generation from iNOS, while ONOO− had little effect. Overall, we observe that ROS and RNS regulate the function of iNOS shifting the balance of NO and production. The dose dependence and reversibility of this process are characterized.
Section snippets
Materials
Rat iNOS was expressed in Escherichia coli as described [27]. Peroxynitrite and degraded peroxynitrite were purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). Tetrahydro-l-biopterin (BH4) was obtained from Cayman Chemical Company. HisTrap affinity column, HiTrap desalting column, Hiload 16/60 Superdex 200, Superdex 200 10/300 GL Tricorn™ high performance chromatography column, and gel filtration calibration kits were purchased from Amersham Pharmacia Biosciences (Pittsburgh,
Oxidant pre-exposure decreases NO production from BH4-replete iNOS
Initial experiments were carried out to directly determine if exposure of BH4-replete iNOS to the four biologically relevant oxidants (ONOO−, OH, H2O2, or ) would alter NO production. For these experiments, we exposed iNOS to a 50-fold molar excess of each oxidant. After each oxidant exposure was allowed to run to completion, or quenched, we used the NO spin trap Fe–MGD and 15N isotopically labeled l-arginine to measure NO derived from the guanidino group of l-arginine, ensuring that all
Discussion
In the absence of l-arginine or with BH4 depletion, production of NO from iNOS becomes uncoupled from the oxidation of NADPH, resulting in prominent generation. This uncoupling of the enzyme with formation would also further shift the balance of NO and present and likely induce cellular injury. However, the precise sensitivity of the coupling of iNOS to specific oxidant stress had not been determined. This study demonstrates that in the absence of additional BH4 both RNS and ROS
Acknowledgment
This work was supported by the National Institutes of Health Grants HL63744, HL65608, and HL38324.
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