Regulation of the Pulmonary Vascular Signaling System by ROS and RNS

Cells are normally protected from the cytotoxic effects of02'~ by SOD, but when elevated, 02" can either be dismutated to produce H202, or it can interact with NO to produce 0N00\ An overproduction of free radicals exert cytotoxic effects, but these oxidants species can also interact with cell-control mechanisms and potentially contribute to signaling processes. As shown in the Figure 1, certain signaling systems are very sensitive to low levels of 02'~ and H202 normally produced by cells, and these mechanisms are likely to participate in physiological regulation. In addition, other signaling systems activated by conditions generating higher levels of oxidants are generally associated with the mechanisms contributing to pathophysiological processes and cellular responses to oxidative stress.

Mechanical changes can influence 02'~ production. For example, alterations in the shear stress caused by increased blood flow promote endothelium-derived 02" generation in the pulmonary circulation (6). Local levels of 02" are also elevated during lung injury or distress conditions by an imbalance between its rate of formation and its rate of removal via SOD. When the concentrations of 02'~ rise over the nanomolar concentration range, it directly interacts with catecholamines (including the neurotransmitter norepinephrine and the hormone epinephrine) and NO, which inactivates the bioactivity of these mediators. 02" inhibits vascular relaxation to H202, which may be a result of 02" inhibiting the activity of catalase.02" inactivates glutathione peroxidase by oxidizing selenium and aconitase by interacting with iron-sulfur center in the enzyme resulting in increased oxidant stress and an impairment of mitochondrial function. Increased levels of 02'" reductively release iron from the iron-sulfur centers and from ferritin, and the reaction of the released iron with other ROS and RNS result in the modulation of other signaling processes or in the initiation of tissue injury. Signaling systems most sensitive to changes in intracellular levels ofH202 are linked to the metabolism of H202 by cyclooxygenase, catalase and GSH peroxidase. Accumulating evidences suggest that H202 increases the levels of oxidized GSH and promotes S-thiolation of proteins (disulfide formation of protein thiols with GSH) that relays intracellular signaling (6, 34-36). A new concept is that endothelium cytochrome P450-derived H202 may function to open channels and induce relaxation through hyperpolarization of the underlying VSM cells through gap junctions.

Figure J. A schematic representation of the interactions of radicals, oxidants and redox changes with the signaling systems. Question marks indicate that the final pathway that changes the channel activity is still uncertain.

It is now well established that endothelium- and perhaps other sources such as smooth muscle- and mitochondrial-derived NO play pivotal roles in the regulation of pulmonary circulation (3, 17, 25). As such, NO is critically important in modulating pulmonary vascular function and inhibiting pulmonary vascular remodeling. NO induces relaxation of PA through activation ofsGC-cGMP pathway and opening of big conductance calcium-activated K+ channels (1). It has also been proposed that NO potentiates the Ca2+ uptake through sarcoplasmic reticulum (SR) Ca2+-ATPase and promotes cGMP-independent relaxation of bovine PA under hypoxic conditions (21). In contrast to micromolar concentrations of H202 which stimulate the release of endothelium NOS-derived NO, 02*- destroys NO, antagonizes endothelium-dependent relaxation, and has been reported to promote contraction of PA due to the formation of ONOO". High levels of ONOO" are cytotoxic, but low concentrations of ONOO" may participate in signaling-like mechanisms including PA relaxation, the inhibition of catalase and H202-elicited cGMP-mediated relaxation of PA, protein tyrosine nitration that may interfere with phosphorylation/dephosphorylation signaling pathways or alter protein functions, and based on studies in skeletal muscle it nitrosylates and inhibits the activity of SR Ca2+-ATPase. Therefore, it is plausible that Po2 governs the oxidative state of NO and modulates the action(s) ofNO on the control of intracellular Ca2+ homeostasis, which ultimately regulates vasomotor tone and blood flow that participate in circulatory Po2-elicited responses. Thus conditions that promote ONOO" formation could influence both acute blood flow responses linked to changes in Po2, and the evolution of pulmonary vascular disease.

The sGC-derived second messenger cGMP is a key regulator of vascular relaxation. Both exogenously and endogenously produced NO and H202 promote relaxation through cGMP-mediated pathways. The H202 appears to activate sGC by two different pathways (Figure 1): i) H202 stimulates the production of NO by NOS in endothelium, and ii) vascular smooth muscle sGC activity is also increased by a mechanism involving the metabolism of H202 by catalase. Stimulation of sGC by the metabolism of peroxide by catalase occurs at high picomolar to low nanomolar concentrations ofH202 and it promotes relaxation of the PA without changes in GSH redox (34). However, the specific pathways through which H202 and cGMP cause relaxation of pulmonary arteries remain to be defined. No one has yet studied in detail the role of ROS or RNS in modulating the protein kinase G pathway (cGMP-dependent protein kinase), although preliminary results have suggested that RNS and hypoxia may modulate protein kinase G activity in PASMC (25). Both OH' and 02'~ inhibit the stimulation of sGC by either NO or H202 (34). In fact endogenously 02" generated originating from NADH oxidase inhibits sGC in bovine PA after inactivation of Cu-Zn SOD with diethyldithiocarbamate by impairing NO-elicited relaxation (34, 36). Thus, ROS have several mechanisms through which they can control the activity of sGC and relaxation ofblood vessels mediated by cGMP.

Vascular phospholipases, including PLA2, PLC, and PLD, are stimulated by ROS. Although the signaling pathways elicited by ROS, which control the activity of phospholipases, are not well understood, there is evidence that cytosolic PLAj is stimulated by H202 in endothelial cells viaphosphorylation by tyrosine kinase-, mitogen-activated protein kinase (MAPK)-, and protein kinase C (PKC)-dependent pathways (34). Low levels of H202and ONOO'stimulate the activities of lipoxygenase and cyclooxygenase enzymes, in contrast high levels of H202 have been suggested to inactivate cyclooxygenase and prostacyclin synthase, and increase thromboxane synthesis (13). The pulmonary vasoconstriction in pig and rabbit lungs caused by 02" appears to be mediated by thromboxane, leukotrienes, and prostaglandins (6). Additionally, NO and ONOO" potentially interact with and regulate the activities of the heme-iron containing cytochrome P450, and the levels of 20-HETE production, which is a dilator of human PA that causes a prominent inhibition of vasoconstriction in lungs elicited by hypoxia (38).

Observations demonstrating the potential importance of oxidant regulation of ion transport mechanisms are slowly accumulating. It has been shown that elevated levels ofH202 cause K+ channel-dependent relaxation of VSM (19, 33, 37). Although the actual relationship between ROS or RNS and the function of cellular ion channels are not well characterized, evidence exists for the potential importance of several processes. A cysteine moiety in the a- or P-subunit of plasma membrane Kv channels have been shown to control the opening and closing of access to the channel by a "ball and chain" mechanism, thereby regulating channel activity. It has been shown that oxidation of the cysteine thiol group by ROS or RNS results in the opening of voltage-gated K+ channels, namely Kvl.l, Kv1.4, Kv1.5, and Kv2.1, leading to hyperpolarization of the plasma membrane and relaxation of PA. Likewise, there are reports suggesting that Ca2+-regulated K+ channels mediate the vasodilation elicited by ROS in the rat pulmonary circulation (32, 33, 37). Oxidation of thiol molecules on the a,-subunit of L-type Ca2+ channel protein by ROS or oxidizing agents also inactivates these channels by modulating voltage-gating and the influx of Ca2+ currents, which would result in relaxation of VSMC. Conversely, inward Ca2+ currents through non-specific cationic channels are increased due to oxidation of thiol groups on the channel proteins by free radicals. Other signaling mechanisms that are regulated by radical species, such as cAMP- and cGMP-dependent processes, control the function of ion channels in protein kinase-dependent or -independent manner. As 02"~ modulates Na+/H+ antiport and Na+/K+ pump activity in cultured pulmonary artery endothelial cells (6), an alteration in ion pump activity could potentially regulate the ion channels on the plasma membranes of these cells. Furthermore, L-type Ca2+ channels are indirectly activated by phosphorylation of the channel protein through PKC, which is known to be stimulated by ROS, and this could cause an increase Ca2+ influx and VSMC dysfunction.

The mechanisms that control the uptake and/or release of SR Ca2+ are also sensitive to oxidants. 02" appears to inhibit the breakdown of inositol 1,4,5-triphosphate and promote the release ofCa2+ from the SR of bovine VSMC. In contrast, although high levels of 02" and H202 selectively disrupt SR Ca2+-ATPase possibly through the irreversible oxidation of thiol groups or by directly attacking the ATP-binding sites, and there is evidence suggesting that 02" augments the up-take ofCa2+ into the SR of bo vine PASMC (6, 36). Thus, ROS induce different effects on the activity of cation channels, which produces diverse responses in VSMC. To add to this complexity arachidonic acid derived from phospholipid breakdown, prostaglandins derived from cyclooxygenase, and 15- and 20-HETE derived from cytochrome P450 following stimulation by ROS or RNS are also potent modulators of ion channel activity. As multiple vascular ion channels are potentially controlled by variety of redox- and oxidant-linked mechanisms, this could likely be the origin of diverse responses of different vascular beds to ROS or RNS.

Redox status of cytosolic NADP(H), NAD(H), and GSH are tightly controlled in vascular and non-vascular cells, and changes in the redox status of these systems are also potentially linked to controlling the expression of redox-regulated signaling mechanisms. It is currently thought that NADPH and GSH are major reducing systems in cells, and that the majority of cytosolic NADPH is maintained in its reduced form by the pentose phosphate pathway. Recent reports suggest that loss of control of NADPH/NADH/GSH redox potential, which is generally associated with ROS metabolism, is one of the important mediators of signaling systems in blood vessels. An example of this mediation are observations that the elevation of NADP7NAD7GSSG in pulmonary and aorta VSMC opens K+ channels and attenuates the release ofCa2+ from the SR associated with causing relaxation of PA and aorta (11). These redox changes may affect ion channel activities by GSSG-mediated S-thiolation of the channel protein or by direct interaction of NADP+/NAD+ with recently discovered nucleotide-binding sites on the Kv 1.4 channel protein (23). Loss of NADPH also inactivates L-type currents in cardiac myocytes and suppresses myocardial contractility (12). In addition, NADPH-linked oxidoreductase systems are involved in the synthesis of NO and in preventing the oxidation of the heme and/or thiols on sGC, inactivating sGC and blocking cGMP-mediated NO-elicited relaxation of pulmonary vessels. NAD(H) redox has also been implicated in the control of 02" production through its role as a substrate for NADH oxidase and interactions with several signaling pathways (34, 36). Thus, the redox status of NADP(H), NAD(H), and GSH is likely to have a major influence on the function of multiple oxidant-associated signaling mechanisms, which regulate vascular function. Imbalances in these redox systems are now emerging as factors, which have serious consequences on the signaling pathways that control vasomotor tone, and alterations in these systems maybe a formidable cause of dysfunction in various pulmonary vascular diseases.

Multiple components ofprotein phosphorylation cascades are altered by ROS and RNS. Modification of an essential thiol at the catalytic site of tyrosine-specific protein phosphatases by either ROS or RNS appears to inhibit the in phosphatase activities, and as a consequence, tyrosine kinase-related signaling pathways becomes activated (34). Thus, the stimulation of tyrosine phosphorylation by H202 activates most forms of PKC in a diacylglycerol-independent manner that leads to stimulation of numerous PKC-dependent vascular signaling pathways. ROS also activate the various MAPK systems that can potentially regulate vascular force generation, proliferation, and adaptive responses to injury (34). In pulmonary arteries, PKC has been reported to mediate vasoconstriction caused by increased 02" (6). Interestingly, a recent study suggested that H202-induced activation of p42/p44 MAPK mediates the force generation of previously stretched PA (36). The p42/p44 MAPK pathway has been shown to stimulate a receptor-activated Ca2+-independent contractile response in VSMC through the phosphorylation of caldesmon (7). Thus, ROS and RNS have multiple ways of interacting with processes that modulate the Ca2+-sensitivity of the contractile apparatus, and augment phosphorylation of myosin light chains. The phosphorylation of proteins involved in the mitogenic processes, which are normally activated by receptor-regulated signaling systems, are also generally enhanced by ROS (24, 34). Some of the important interactions of ROS with signaling pathways that are potentially involved in regulating PA function during HPV are discussed in the following sections.

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