There are multiple oxidase enzymes that generate ROS, and each enzyme appears to have specific mechanisms that regulate ROS production and specific cellular localization sites. Evidence is rapidly emerging that one of the most important types of oxidases in physiological processes are
NAD(P)H oxidases (containing specific nox subunits) that resemble the oxidase initially identified in phagocytic cells, which is now called nox-2. Vascular smooth muscle and endothelium appears to contain the nox-1, nox-2 and nox-4 forms of these nox oxidases. The nox oxidases appear to be composed of membrane bound nox and p22phox subunits possessing an electron transport system that transfers electrons from cytosolic NADPH and/or NADH to molecular oxygen through a flavin site and a 6558-type cytochrome. Electron transfer resulting in the generation of ROS by the nox oxidases appears to be controlled by stimulation of the binding of one or more cytosolic protein subunits that include or resemble rac-1, p47phox and p67phox. All cells seem to generate ROS under unstimulated conditions, and it appears that nox-containing systems are an important contributor to the basal levels of ROS production in vascular cells. Stimuli including stretch and shear and the activation of receptors linked to cell growth (e.g., angiotensin II) are thought to increase nox activity through cytosolic subunit binding triggered by mechanisms including phosphory-lation by protein kinase C or other signaling processes. Some growth factors also appear to increase oxidase activity through promoting the expression of the nox and p22phox subunits. The nox enzymes that are present in vascular cells appear to have important roles in intracellular signaling events that control vascular function and cellular growth.
Endothelium contains multiple additional oxidant-generating enzyme systems, including cytochrome P450, NO synthase (NOS), cyclooxygenase (COX), and xanthine oxidase (XO). Recent evidence suggests that H2O2 is an important endothelium-derived relaxing factor, and receptor stimulation of its generation by cytochrome P450 and other oxidases appears to be a source of its generation. Although the major product of the NOS reaction is NO, this system has NADPH oxidase activity that is suppressed by the availability of the reduced form of its cofactor tetrahydro-biopterin, and by the availability of L-arginine for NO biosynthesis. When NOS is making both NO and O2-, the reaction between these molecules generates peroxynitrite (ONOO-), which is a reactive species that generates other RNS (including nitrogen dioxide), which oxidizes tetrahy-drobiopterin and causes thiol oxidation, nitrosation and nitration, and tyrosine nitration, whereas, when the generation of O2- is the primary product of NOS, this system appears to become a major source of vasoactive endothe-lium-derived H2O2 generation. Vascular disease processes are known to promote oxidant production from NOS, COX, and XO. The generation of O2- from COX seems to be associated with the availability high levels of arachidonic acid and high rates of prostaglandin generation by this enzyme. Generation of both O2- and H2O2 by XO appears to require conversion of this enzyme to an oxidase from its dehydro-genase form by a combination of thiol oxidation and/or pro-teolysis, and the availability of its substrates hypoxanthine and xanthine. Prolonged hypoxia and reoxygenation generally stimulates XO activation and the accumulation of its substrates from the degradation of tissue ATP. Thus, while endothelium is a source of vasoactive levels of H2O2, this cell type can also become a major source of vascular oxidant production during pathophysiological processes.
Mitochondria of vascular cells also appear to have an important role in controlling oxidant generation either through producing O2- or as a result of their role in controlling the redox status of cytosolic NAD(H), and perhaps NADP(H). The NADH dehydrogenase and coenzyme Q sites in the electron transport chain appear to be sites where electrons can be transferred to molecular oxygen, and processes associated with the development of apoptosis seem to markedly increase mitochondrial oxidant generation. Mitochondria appear to contribute to the sensing of physiological PO2 levels through poorly understood redox processes that seem to be linked to the generation of ROS.
Mechanisms of Interactions of Oxidant Species with Vascular Signaling Systems
Each oxidant species has unique ways, shown in Figure 1, of interacting with the signaling systems listed in Table I as a result of its chemical and metabolic properties.
The major ROS that is generated by cellular oxidases appears to be O2-, which is a negatively charged free radical at physiological pH, with very selective chemical reaction properties. The levels of SOD in the cytosol (SOD-1) and mitochondria (SOD-2) appear to function in a manner that maintains intracellular O2- concentrations in the picomolar
Table I Oxidant Species and Some of Their Interaction with Signaling Systems.
Cyclooxygenase Glutathione peroxidase Catalase Fe2+
Thiols (RSH) Mitochondria (Fe-S) Tyrosine
Oxidation or Nitrosation (RSSG, RSSR, RSOH, RSNOx)
Inhibits actions of NO
Releases iron bound to proteins as Fe2+
Converts O2- to H2O2
Stimulates prostaglandin production
Converts GSH to GSSG
Stimulates sGC and cGMP production
Generates reactive "OH" species
Stimulates sGC and cGMP production Reversibly inhibits mitochondrial respiration Generates peroxynitrite (ONOO) Oxidation and nitrosation Irreversible inhibition of respiration Nitration, inactivation of PGI2 synthase
Opens potassium channels Activates tyrosine phosphorylation Altered Ca2+ reuptake mechanisms Inhibits calcium influx range. These low levels of O2- appear to keep it from directly interacting with most signaling systems. Because of the extremely rapid rate of reaction of NO with O2-, increases in O2- are observed to readily attenuate physiological actions of NO [e.g., stimulation of soluble guanylate cyclase (sGC) and inhibition of tissue mitochondrial respiration] and can form ONOO- in amounts that interact with signaling systems when high levels of NO are present. Vascular smooth muscle secrete an extracellular form of SOD (SOD-3), which appears to protect NO from O2- as it diffuses through the vessel wall. Elevated levels of O2- are also known to damage iron-sulfur centers, associated with the inhibition of mitochondrial respiration, and release of iron. A major role for O2- and SOD in signaling appears to be the generation of H2O2.
Hydrogen peroxide has a vast array of interactions with signaling systems, and its basal levels in cells are thought to be in the low nanomolar range. The signaling mechanisms most sensitive to the actions of H2O2 appear to be linked to its metabolism by heme peroxidases, catalase, and glu-tathione (GSH) peroxidase, and these systems participate in the generation of prostaglandins, cGMP, and oxidized glutathione (GSSG), respectively. The local concentration of GSSG and proteins present that have thiol groups (RSH) that can be S-thiolated (RS-SG) or oxidized to disulfides (RSSR) are key processes that link ROS to cellular signaling systems. Some of the cellular proteins and systems known to be regulated by thiol redox that potentially influence microvascular function are included in Table I. When ferrous iron (Fe2+) is released, it promotes the formation of very reactive or hydroxyl radical ("•OH")-like species from H2O2, which cause cellular injury.
Evidence is emerging that RNS have important regulatory functions through interactions with signaling systems. These species readily modify proteins with reactive thiol groups (see Table I) by nitrosating (RS-NO), nitrating (RS-NO2), or oxidizing (RS-SG, RSSR, RSOH, RSOx) them in a manner similar to H2O2 (or elevated levels of other ROS). Protein tyrosine groups, iron-sulfur centers, and unsaturated fatty acids are also readily modified by RNS (and ROS). Some well-documented actions of ONOO- that potentially influence microvascular regulation include the inactivation of prostaglandin I2 synthase by nitration of a key active-site tyrosine, and inactivation of tissue mitochondrial respiration by damaging key iron-sulfur centers. Thiols linked to zinc binding sites are also very sensitive to oxidative processes that release zinc and promote cellular signaling. While O2-and peroxide have the potential for interactions with the protein thiols and other sites modified by RNS, there seems to be a need for an enhancement of the reactivity of the thiol site (e.g., by acidification) or ROS by cofactors such as iron for the observance of selective signaling-type regulation. Thus, although both peroxide metabolism and RNS cause the oxidation of GSH to GSSG, the chemical reactive properties that RNS possess enable them to have multiple additional regulatory and pathophysiological interactions, especially at activated thiol and metal-binding sites.
Tyrosine phosphorylation, potassium channel opening, and certain systems that control intracellular Ca2+ and phospholipid metabolism appear to be cellular regulatory systems having components that seem to be directly regulated by ROS, RNS, or changes in the redox status of cytosolic NADH, NADPH, and/or GSH. Tyrosine phosphatases have activated thiols (RSH) at their catalytic sites that appear to be readily modified or oxidized by ROS and RNS, resulting in inhibition of the tyrosine-phosphate hydrolyzing activity of these enzymes. Some tyrosine kinases appear to autoacti-vate when the status of their phosphorylated tyrosine groups changes. Tyrosine phosphorylation appears to control multiple processes often associated with vascular contraction, gene expression, and cell growth through changes in the activities of systems including protein kinases B and C, growth factor receptor signaling, and mitogen-activated protein (MAP) kinases. Potassium channels often open when they undergo oxidation, and the thiol groups they contain may be key sites for redox regulation. Plasma membrane Ca2+ channels (e.g., L-type channels) and the sarcoplasmic-endoplasmic reticulum Ca2+-ATP pump or SERCA also appear to readily show inhibition of Ca2+ influx and alterations in Ca2+ reuptake, respectively, when these systems are modified by thiol oxidation. Many of these oxidant-regulated signaling systems seem to function in a coordinated manner to control phospholipid signaling, including the release of arachidonic acid, which is used for the biosynthesis of prostaglandins. The hyperpolarization-mediated relaxation induced by opening K+ channels activated by H2O2 has resulted in it being considered as an endothelium-derived hyperpolarizing factor. Overall, the subcellular localization of both ROS generation and redox changes observed may have fundamental roles in controlling which oxidant-regulated signaling mechanisms are activated, and which physiological or pathophysiological responses are observed.
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