The Role of Reactive Oxygen and Nitrogen Species in the Expression and Splicing of Nitric Oxide Receptor

Abstract

Significance:

Nitric oxide (NO)-dependent signaling is critical to many cellular functions and physiological processes. Soluble guanylyl cyclase (sGC) acts as an NO receptor and mediates the majority of NO functions. The signaling between NO and sGC is strongly altered by reactive oxygen and nitrogen species.

Recent Advances:

Besides NO scavenging, sGC is affected by oxidation/loss of sGC heme, oxidation, or nitrosation of cysteine residues and phosphorylation. Apo-sGC or sGC containing oxidized heme is targeted for degradation. sGC transcription and the stability of sGC mRNA are also affected by oxidative stress.

Critical Issues:

Studies cited in this review suggest the existence of compensatory processes that adapt cellular processes to diminished sGC function under conditions of short-term or moderate oxidative stress. Alternative splicing of sGC transcripts is discussed as a mechanism with the potential to both enhance and reduce sGC function. The expression of α1 isoform B, a functional and stable splice variant of human α1 sGC subunit, is proposed as one of such compensatory mechanisms. The expression of dysfunctional splice isoforms is discussed as a contributor to decreased sGC function in vascular disease.

Future Directions:

Targeting the process of sGC splicing may be an important approach to maintain the composition of sGC transcripts that are expressed in healthy tissues under normal conditions. Emerging new strategies that allow for targeted manipulations of RNA splicing offer opportunities to use this approach as a preventive measure and to control the composition of sGC splice isoforms. Rational management of expressed sGC splice forms may be a valuable complementary treatment strategy for existing sGC-directed therapies. Antioxid. Redox Signal. 26, 122–136.

Introduction

Since the recognition of nitric oxide (NO) as the endothelial-derived relaxing factor in the late 1970s to early 1980s, our understanding of NO as a fundamental signaling molecule expanded tremendously. It is now well accepted that NO-dependent signaling affects virtually every cellular function and a plethora of physiological processes. This small cell-permeable molecule is an essential player, or at least an important modulator, in processes as diverse as neurotransmission, vascular function, platelet and leukocyte adhesion, thrombogenesis, cellular respiration, apoptosis, mitochondrial biogenesis, cell migration or proliferation, and so forth. A number of excellent reviews were written on these subjects (27, 35, 82, 114, 119).

Most of the physiological effects of NO are mediated by the NO-sensitive soluble guanylyl cyclase (GTP pyrophospate-lyase, sGC). sGC is an enzyme-linked receptor for NO that is composed of one α and one β subunit. Two separate genes for each subunit (α1, α2, β1, and β2) are found in mammals, but only the ubiquitously expressed α1β1 and the less abundant α2β1 heterodimers have been detected in vivo (71). The domain organization of most abundant α1 and β1 subunits is depicted in Figure 1. The C-terminal half of both subunits is necessary and sufficient to form a functional catalytic site with low cyclic guanosine monophosphate (cGMP)-forming activity (24). The NO receptor portion of sGC is located in the N-terminal region of the enzyme (25). Although the function of the N-terminal domain of the α subunit is not entirely understood, it is clear that the N-terminal domain of the β subunit is essential in harboring the ferrous heme prosthetic group. It is to this heme moiety that NO binds with a high affinity.

FIG. 1.

Domain organization of sGC α1 and β1 splice variants. The underlined numbers above bars are domain boundaries. The numbering is with respect to canonical variants of α1 and β1 sGC. The size and positions of splice-variant specific inserts (black boxes) are indicated. The names of splice transcripts and proteins are in accordance with updated NCBI nomenclature. Parentheses list previously used names for the splice forms. CAT, catalytic domain; CC, coiled coil domain; HNOX, domain binding heme/NO/oxygen; NO, nitric oxide; PAS, Per/Arnt/Sim domain; sGC, soluble guanylyl cyclase.

Although the sGC–heme complex has many features that are similar to the heme–hemoglobin complex (i.e., a ferrous heme coordinated by a histidine residue), sGC exhibits an exceptional selectivity toward NO. Both functional (35) and direct binding (63, 126) studies demonstrate that sGC binds NO with a K _D of ∼50 nM. Moreover, the binding is very fast and reaches a diffusion limited rate (139). On the other hand, sGC does not bind oxygen at physiologic pressure and concentrations (128), whereas the 250 μM K _D for carbon monoxide (CO) (62) is well outside of the physiological range of CO.

The ferrous status of sGC heme is essential for NO sensing, since in the ferric form the affinity for NO is very low (128). The binding of NO to sGC heme triggers a number of conformational changes that eventually result in several hundred-fold enhancements of the cGMP-forming activity in the catalytic region of sGC (53). The exact nature of these conformational changes is currently the subject of intense investigation (14, 16, 59, 105, 130). This set of properties makes sGC uniquely sensitive to transient low nanomolar concentrations of NO that are typically produced under normal physiologic conditions (40). This ligand binding step is then amplified through the synthesis of the secondary messenger cGMP. However, these very same properties make the NO–sGC communication very susceptible to the effects of various reactive oxygen species (ROS).

Ever since the early reports suggested that sGC-dependent synthesis of cGMP may be activated by hydrogen peroxide (H₂O₂) (135) or superoxide dismutase (SOD) (75), the complexity of the relationship between NO-dependent activation of sGC and ROS was recognized. The term Reactive Oxygen Species is rather amorphous and is generally used in reference to the initial species that are generated by oxygen reduction (superoxide and H₂O₂) and their secondary reaction products. Some of these ROS have the capacity to directly react with NO with various rates and to form reactive nitrogen species (RNS). Among ROS, superoxide anion (O₂ ⁻) has the highest reaction rate with NO (84). In fact, the reaction between superoxide anion and NO is up to 100 times faster than the interaction between NO and sGC (84, 127). Therefore, under oxidative stress, when the level of generated superoxide exceeds the capacity of SODs to inactivate it, the bioavailabilty of NO (and therefore activation of sGC) is reduced, whereas the level of peroxynitrite increases.

Although it is generally understood that ROS is not a single entity, but a conglomerate of species with various redox potentials, different specificities and reaction rates, it is often impossible to discern the specific agent that is the major contributor to the investigated biological effect (30, 137). With such stipulation, this review attempts to catalog the effects of major ROS and RNS on sGC activity and expression.

As described next, cellular oxidative conditions and elevated ROS/RNS not only affect the bioavailability of NO but also, in many instances, affect the activity of sGC or change the redox status of sGC heme. Besides these direct effects on sGC function, ROS and RNS cause post-translational modifications, facilitate sGC degradation, affect the synthesis or stability of sGC mRNA, and alter the composition of expressed variants of sGC splice forms. This article provides a concise overview of some of the known effects of oxidative stress and ROS on sGC function and expression, with special emphasis on the regulation of sGC splicing.

ROS/RNS and sGC

Molecular oxygen has been recognized since the 1950s as a factor that greatly amplifies the toxicity from radiation-dependent cellular damage. The discovery of SOD class of enzymes (51, 91) that possess the ability to scavenge the superoxide provided the first evidence that oxygen-derived free radicals such as O₂ ⁻, H₂O₂, and hydroxyl radical (^•OH) may be responsible for the toxicity of oxygen. Hydroxyl radical (^•OH) is, by far, the most active among these three. However, the reactions that generate ^•OH from H₂O₂ via Fenton chemistry are slow, whereas deactivation through interaction with virtually any biological molecule is very fast. The current view is that this combination of factors makes the distance traveled by ^•OH very small, therefore diminishing its biological impact on the cell (84, 137). O₂ ⁻ and H₂O₂ are more stable, and the diffusion rates for these agents are more suitable for the creation of a larger and more lasting oxidative environment (58, 81, 117).

Effects of O₂ ⁻

The effect of O₂ ⁻ on sGC activity appears to be highly dependent on the mode that has been applied to generate or modulate the level of O₂ ⁻. For example, when rat aorta was exposed to O₂ ⁻ generated by xantine/xantine oxidase (X/XO) reaction, the activity of sGC in the aortic lysate (133) was not affected. However, when endogenous generation of superoxide was induced in platelets (78) or rat aortic rings (52), NO-dependent activation of sGC decreased. However, such experiments are opened to interpretation due to possible interference from other oxidative species or through interaction with endogenous NO. Therefore, more conclusive results were sought when sGC-enriched fractions from the lysates of human platelets (10) or purified recombinant sGC (77) were exposed to O₂ ⁻ generated by the X/XO system. In both reports, basal and NO-dependent sGC activities were attenuated. Moreover, O₂ ⁻ inhibited the activation of sGC by the NO-independent stimulator YC-1 (77). Interestingly, Brune et al. observed that the heme-free sGC was also inhibited by O₂ ⁻ (10) and suggested that the inhibitory effect of O₂ ⁻ is not related to changes in the heme status of sGC.

Using an opposite approach, Friebe and colleagues demonstrated that addition of SOD to sGC preparation results in enhanced sGC activity, even without the addition of NO (32). They demonstrated that SOD-mediated stimulation of sGC is due to the elimination of NO scavenging via the reaction with superoxide. In contrast to studies by Brune et al. (10), the SOD-dependent stimulation was not observed with the heme-deficient enzyme.

Effects of ONOO⁻

As mentioned earlier, when O₂ ⁻ levels overwhelm the scavenging capacity of SODs, the reaction between O₂ ⁻ and NO generates peroxynitrite (ONOO⁻), a highly reactive oxidant. Under physiological redox conditions, ONOO⁻ is efficiently inactivated by reactions with cellular thiols, such as glutathione (93). However, under oxidative stress, when the reducing capacity of cellular thiols is exhausted, ONOO⁻ causes substantial oxidative damage and leads to lipid oxidation, DNA damage, protein nitration, and so forth (22, 84).

Interestingly, two rather opposite effects of peroxynitrite on the function of sGC are reported.

A substantial number of studies demonstrate that ONOO⁻ negatively affects sGC activity and function. Exposure of rat aortic rings to SIN-1 was shown to impair endothelium- and NO-dependent vasorelaxation of isolated blood vessels (54, 116). Pre-treatment with ONOO⁻ was also reported to inhibit NO-dependent activation of sGC. At the same time, the vasodilation of rat aorta in response to BAY58-2667 (116) or of rat iliac arteries in response to related BAY60-2770 (123) was enhanced after ONOO⁻ treatment. Since both BAY58-2667 and BAY60-2770 are NO- and heme-independent activators of apo-sGC or sGC with oxidized heme, these studies clearly indicate that oxidation (or loss) of sGC heme takes place after exposure to ONOO⁻. Moreover, Stasch et al. demonstrated that exposure of endothelial cells to ONOO⁻ not only decreased NO-induced cGMP synthesis but also diminished the level of sGC protein (116). The studies by Weber et al. (133) demonstrated that 4-h incubation of rat aorta with a flux of 0.13 μM/min of exogenous ONOO⁻ had no effect on sGC-dependent cGMP synthesis. Diminished NO-dependent cGMP synthesis was observed only when aortas were incubated and when there was a much higher flux of 7.5 μM/min ONOO⁻ (31, 133).

On the other hand, several studies indicated that ONOO⁻ may stimulate sGC activity and function. Collaborative studies by Mayer et al. reported that in the presence of glutathione ONOO⁻ acts as a potent sGC activator at a concentration above 10 μM (68, 69). The activation of sGC was not observed in the absence of glutathione (68, 69). A similar dependency on glutathione was observed when sGC activity was measured in endothelial cells exposed to SIN-1, a dual donor of NO and O₂ ⁻ (69). The authors suggested that formation of nitrosothiol is an essential step in activation of sGC by peroxynitrite. This conclusion was independently corroborated by Wolin's group, which demonstrated that bovine coronary and pulmonary arteries relax in response to ONOO⁻ (43, 138). They also detected the generation of NO and nitrosothiols from ONOO⁻ when incubated with aortic tissues or glutathione-containing buffer. It should be noted that in these studies, the consequences of ONOO- treatment on NO-dependent activation of sGC was not evaluated.

The contrast between these two different sets of studies may reflect the biphasic effect of NOO⁻ on sGC. When the pool of cellular thiols is high, an acute (several minutes) increase in ONOO⁻ may be accompanied by its conversion into NO or nitrosothiols, resulting in a short-term activation of sGC (43, 68, 69, 138). However, sustained fluxes of high ONOO⁻ are typically generated under inflammatory conditions, when the pool of intracellular thiols is usually depleted. It is reasonable to assume that under such conditions, longer (>0.5 h) exposure to exogenous ONOO⁻ (54, 116, 133) or chronic generation of endogenous ONOO⁻ under inflammatory conditions and/or vascular dysfunctions (116) causes oxidation of sGC heme and impaired sGC function.

Effect of sGC nitrosation

Elevated cellular oxidative stress is coupled with a higher content of RNS. Moreover, chronic administration of some nitrovasodilators, such as nitroglycerin, was shown to increase both oxidative and nitrosative stress (80, 89). An earlier study by Waldman et al. suggested that desensitization of sGC from thoracic aorta or human coronary artery to nitrovasodilators is due to a relatively stable molecular alteration of sGC that makes the enzyme specifically less sensitive to NO, but does not change the response to NO-independent activation by the heme-replacing protoporphyrin IX (131). Sayed et al. were the first to test whether nitrosation of sGC cysteines accounts for sGC desensitization. They demonstrated S-nitrosated sGC in primary aortic smooth muscle cells (SMCs) exposed to S-nitrosocysteine, in human umbilical vein endothelial cells treated with vascular endothelial growth factor, and in isolated aorta after sustained exposure to acetylcholine (97). These studies demonstrated that S-nitrosated sGC has impaired NO-stimulated activity and suggested the involvement of cysteine 243 in α1 and cysteine 122 in β1 sGC. In a later study, S-nitrosation of sGC was directly linked to the development of tolerance to nitroglycerin (98).

Accumulation of nitrosated β1 sGC was also reported in vivo in transgenic mice that overexpress eNOS in endothelial cells (83). In this article, nitrosation also resulted in lower sGC activation by NO in lungs. It should be pointed out that such a loss of sGC activity was observed only in the lungs, where the higher oxygenation is more permissive for generation of RNS. Mayer et al. also reported partial inactivation of sGC by stoichiometric S-nitrosation, when S-nitrosating agent dinitrosyl iron complexes (DNIC) were used (67). These authors also emphasized the critical role of cellular thiols in the outcome of sGC response to S-nitrosating agents. They demonstrated that in the presence of glutathione DNIC act as sGC activators, whereas in the absence of glutathione S-nitrosation desensitizes sGC. This study clearly indicates that the state of the cellular thiol pool, which is rapidly exhausted under oxidative conditions, predetermines the outcome of the nitrosative pressure on sGC function.

Effect of H₂O₂

Biologically generated H₂O₂ is predominantly the product of superoxide deactivation. SOD-catalyzed breakdown of O₂ ⁻ is the primary mean of H₂O₂ synthesis, with oxidation of O₂ ⁻ by reduced iron/sulfur clusters playing a secondary, but measurable, role (137). Although H₂O₂ is a two-electron oxidant, it usually reacts poorly with most biological molecules. The most common reactions with participation of H₂O₂ are the oxidation of cellular and protein thiols and the formation of hydroxyl radicals via Fenton chemistry. Interestingly, sGC contains a large number of cysteines that could be potential targets for H₂O_2. Moreover, sGC heme contains a coordinated ferrous iron that can potentially catalyze the Haber–Weiss conversion of H₂O₂ into hydroxyl ion and ^•OH (46).

Generation of ^•OH should result in oxidative damage to the immediate vicinity of the sGC heme pocket and oxidation of heme. Surprisingly, despite such apparently favorable conditions for sGC damage by H₂O₂, there is little evidence that such damage takes place. In fact, the opposite is true. Since White et al. reported that H₂O₂ elevates sGC activity in lung homogenates (135), a substantial number of reports described a rather complex relationship between sGC activity and expression and H₂O₂ exposure. Endothelium-independent sGC-dependent relaxation to H₂O₂ has been extensively documented by using bovine pulmonary or coronary arteries (12, 13, 43). This relaxation cannot be attributed exclusively to the disulfide linking, dimerization, and activation of PKGIα caused by H₂O₂ (11), since elevated cGMP production in isolated blood vessels after exposure to H₂O₂ has been reported (12, 13). The mechanistic details of H₂O₂-dependent activation of sGC are not entirely understood, but the participation of catalase has been proposed. The compound I species of catalase has been postulated as the crucial component for sGC activation (12, 13, 17). Oxidation of sGC heme does not appear to be essential for H₂O₂-dependent activation of sGC, since administration of ODQ, a specific sGC heme oxidant, did not substantially alter the relaxation of bovine arteries induced by H₂O₂ (43).

A number of reports indicate that the potential benefit of sGC-dependent vasorelaxation in response to H₂O₂ is limited due to the decrease of bioavailable NO caused by H₂O₂. Diminished NO-dependent vasorelaxation after the treatment with exogenous H₂O₂ was reported for rabbit aortic rings (124) and denuded rat iliac arteries (122). Diminished NO-dependent cGMP synthesis after exposure to H₂O₂ was also recorded in various models, including primary cultures of rat aortic SMC (36), rat pulmonary artery endothelial cells (EC) (124), or SMCs of fetal lamb pulmonary arteries (134). At the same time, some studies showed that H₂O₂ does not alter cGMP synthesis in response to BAY41-2272 (122, 124) or BAY60-2770 (122), supporting the notion that the redox state of sGC is not affected by H₂O₂.

Post-translational modifications of sGC may also be important in mediating the effect of H₂O₂. Treatment with H₂O₂ of PC12 cells, rat aortic SMCs, and rat aortic tissue caused phosphorylation of the Tyr192 of the β1 sGC subunit (72). Finally, prolonged exposure to H₂O₂ affects the levels of sGC protein and mRNA, as described further in this review.

Effect of complex oxidative stress on sGC function

NO/sGC/cGMP signaling is essential for various cardiovascular processes, many of which are directly affected by ROS. For example, ROS negatively affect the functions of smooth muscle, their growth, differentiation, and death (44, 118). ROS are also regarded as a risk factor for many cardiovascular disorders. Increased scavenging of NO due to inactivation by O₂ ⁻ and generation of peroxynitrite is a strong contributing factor to vascular dysfunctions. However, the dysfunctions of NO/cGMP signaling in pathological conditions can only partially be attributed to the scavenging of NO. As discussed next, ROS directly affect the function and redox state of sGC and alter sGC expression.

Substantial progress in the understanding of sGC status under conditions of oxidative stress was made after the development of heme-independent sGC activators, such as BAY58-2667 (113) or HMR 1766 (101). These compounds strongly activate sGC, which lacks heme or has an oxidized ferric heme (102, 115). Therefore, the combination of NO donors and/or heme-independent activators provides a comprehensive probing of the status of sGC heme in cells and tissues from animals with various conditions that are associated with elevated oxidative stress. This type of analysis revealed that oxidation of sGC heme is a common response in many pathological conditions. Diminished NO response that coincides with elevated response to heme-independent activators was observed in aorta from hyperlipidemic watanabe heritable hyperlipidemic (WHHL) rabbits, mesocolon arteries from type II diabetic patients, and aortic rings from spontaneously hypertensive rats (SHR) (116). A similar response was observed ex vivo with primary rat vascular smooth muscle cells (VSMCs), in which oxidative stress was caused by menadione or rotenone (140), or in monkey coronary arteries after hypoxia or hypoxia/reoxygenation injury (121).

Heme oxidation is not the only change in sGC status that is reported by various groups. Multiple reports indicate that sGC thiol groups are also targeted during oxidative conditions. For example, chronic exposure to aldosterone of bovine VSMC or COS-7 cells expressing sGC affects NO-induced cGMP synthesis and decreases sGC activity, presumably due to the higher level of O₂ ⁻ generated by NADPH oxidase. Proteomic analysis identified a specific peptide of β1 containing various degrees of oxidation of the β1-Cys122 residue (60). Nitrosation of sGC cysteine is another post-translational modification that is often encountered under oxidative conditions. Elevated sGC nitrosation was reported in rats with hypertension caused by chronic AngII administration or rat A7r5 SMC line exposed to AngII (21). sGC nitrosation was also reported in HUVECs treated by vascular endothelial growth factor (VEGF), or in rat aorta after sustained exposure to acetylcholine (97). With the exception of sustained treatment to acetylcholine, these experimental models are not associated with a substantial increase in NO production.

Effect of ROS/RNS on sGC expression

As describe earlier, a diminished response of sGC to NO or heme-dependent stimulators and an elevated response to heme-independent sGC activators are commonly reported effects of ROS or RNS on sGC function. Another often-reported effect observed under various pathological conditions or experimental models of oxidative and nitrosative stress is the decreased level of sGC protein in affected tissues or treated cells.

Diminished levels of sGC subunits were reported in cell culture after treatments with a number of pro-inflammatory agents (3, 85, 90, 103, 120). Lower levels of sGC protein were also recorded after chronic exposure of rat VSMCs (29, 86) or endothelial cells (116) to NO donors. The treatment of isolated rat aorta or cultured VSMC with a wide spectrum of ROS-generating systems (H₂O₂, O₂ ⁻ via XO or menadione sodium bisulphate) reduced the levels of both sGC subunits (36). Diminished sGC protein is also consistently reported in various cardiovascular samples of lean (5, 92, 96) and obese (45) SHR. Multiple studies demonstrate that the hypertensive phenotype of SHR animals is associated with an increased level of ROS. It has been also proposed that an age-dependent increase of ROS, especially O₂ ⁻, is responsible for the lower level of sGC in corpus cavernosum of aging rats (111). The expression of β1 sGC was diminished in rat aorta at late stages of experimental heart failure (6), which was most likely caused by an elevated level of O₂ ⁻ and could be partially reversed by the anti-oxidant action of vitamin E.

At least two different mechanisms have been implicated in the loss of sGC protein under oxidative/nitrosative conditions. Meuer and colleagues demonstrated that ubiquitin-dependent degradation is an important mechanism in controlling sGC level (73). They reported that oxidation of sGC by ODQ in rat aortic samples and cultured cells leads to robust ubiquitination of sGC followed by degradation via the proteasomal pathway. It is not entirely clear whether oxidized or heme-deficient sGC is preferentially ubiquitinylated. In vitro studies performed with purified sGC from Manduca sexta demonstrated that ferric sGC heme is not as tightly bound as ferrous heme and therefore may be lost soon after oxidation (33). Recent studies by Thoonen et al. provided additional support to the notion that cellular heme-deficient sGC is not stable (125). They reported that the Apo-sGC mouse strain expressing heme-free sGC is expressed at a lower level than the wild-type or heterozygote controls. Studies performed by Ghosh et al. (37, 38) demonstrated the importance of heat shock protein 90 (Hsp90) for the proper heme insertion during maturation of sGC. Interestingly, an earlier study reported proteasomal-dependent degradation of sGC in cells treated with Hsp90 inhibitors radicicol and geldanamycin (88). Accumulation of misfolded sGC lacking heme is most likely the reason for sGC degradation in case of Hsp90 deficiency and may be the mechanism of sGC degradation under oxidative stress.

Another likely mechanism of sGC protein reduction is associated with the decreased level of sGC transcripts under various oxidative/nitrosative conditions. It was reported that chronic exposure to NO donors diminishes the level of sGC transcript in rat pulmonary artery SMCs (29, 104) or rat medullary interstitial cells (129). Isolated rat aorta and cultured VSMC treated with ROS-generating agents reduced the steady-state levels of sGC mRNA (36). The lowering of sGC mRNA level is not restricted only to in vitro systems. For example, the level of sGC transcripts was significantly diminished in SHR animals as compared with control rats (96), especially at advanced age (47).

The exact mechanisms that contribute to decreased steady-state level of sGC transcript are only starting to emerge. The work by Gerassimou et al. suggests that the transcription of sGC genes may be directly affected by oxidative stress. The authors demonstrated that the extent of reduction of the α1 sGC transcript in response to H₂O₂ treatment matched very well the reduction of α1 promoter-driven transcription (36). Previous studies of sGC promoter function revealed a number of transcription factors that are essential for regulation of basal sGC expression (61, 109, 110). Although NFκB and CCAAT-BF are particularly important in regulating sGC expression under inflammatory conditions, it remains to be determined whether the same factors are responsible for lower transcription of α1 gene in conditions of oxidative stress.

A number of studies also demonstrated that processes that are responsible for stabilization of sGC mRNA are essential for regulation of the steady-state level of sGC mRNA and may be directly affected by oxidative stress. A series of studies by Kloss and colleagues revealed that human-antigen R (HuR) protein binds the adenine- and uracil-rich elements that are located in the 3′-untranslated regions (UTR) of α1 and β1 sGC transcripts and contributes to stabilization of sGC transcript. It has been demonstrated that decreased HuR levels increase the rate of α1 and β1 mRNAs degradation in rat aortic cells (48, 50). In SHRs, the reduction of HuR protein is associated with decreased α1 and β1 sGC mRNA and contributes to the development of age-dependent hypertension (49). Priviero et al. also correlated decreased sGC activity and protein in mesenteric arteries of SHR animals with a lower level of HuR (92). A decreased level of HuR in murine pulmonary VSMCs cultured after chronic hypoxia was accompanied by the reduction of α1 sGC mRNA (23).

Multiple studies demonstrated that the level of HuR protein changes under oxidative conditions and downregulates the steady-state level of different target mRNA (1, 26). The currently proposed mechanism suggests that thiol-dependent redox regulation of transcription factor activator protein 1 (AP-1), which occurs under oxidative stress, decreases the expression of HuR. This, in turn, destabilized mRNAs targeted by HuR protein (74).

Reduced sGC expression was described in response to O₂ ⁻; H₂O₂, chronic exposure to NO donors, and nitrosative agents, as well as in different animal models associated with increased oxidative stress. However, a number of reports document increased expression of sGC under certain conditions. For example, Kober et al. reported that short-term exposure to O₂ ⁻ (X/XO system) resulted in increased expression of sGC protein in freshly isolated rat aortic rings (52). Increased expression of sGC protein was also observed in cholesterol-fed rabbits (57) and in rats and rabbits after a 3-day administration of nitroglycerine (79). Martin-Garrido et al. reported that exposure of rat aorta and of cultured rat VSMC to H₂O₂ increased the expression of β1, but not α1 protein via the stabilization of β1 transcript due to higher level of HuR (65).

These apparently contradictory observations are, at least in part, explained by the studies of sGC expression in aorta of rats with experimentally induced heart failure. Bauersachs et al. demonstrated that 8 weeks after heart failure was induced, Ach-dependent vasoresponse was diminished, whereas sGC expression was increased (4). However, such a compensatory increase in sGC expression was not observed after 12 weeks of chronic heart failure (6) and a lower level of β1 sGC was reported. These studies suggest that under moderate or short-term oxidative pressure, sGC dysfunction may be compensated by increased sGC expression. Longer or more damaging oxidative stress leads to lower expression of sGC gene and increased degradation of damaged sGC.

To summarize, downregulation of sGC expression and activity under oxidative stress or chronic vascular conditions include inhibition of transcription (3, 87, 90, 104, 120), destabilization of mRNA (49), and protein destabilization in an increased oxidative environment (7, 19, 28). Moderate or short-term oxidative stress may induce a compensatory mechanism, which remains to be fully understood.

sGC Splice Variants and Oxidative Stress

Alternative splicing of sGC transcripts and expression of alternative splice isoforms of sGC emerge as important mechanisms that contribute to the regulation of sGC expression in conditions of oxidative stress. It is estimated that more than 60% of human genes undergo alternative splicing, making it one of the most important post-transcriptional regulatory mechanisms (132). Due to alternative splicing, the same precursor RNA generates different transcripts via incorporation or exclusion of various exon sequences. Variations in UTRs may affect the stability and/or processing of individual transcripts via introduction or elimination of binding sites for regulatory proteins. On the other hand, changes in the coding sequence induced by mRNA splicing may generate polypeptide products with altered functional properties. A number of published reports demonstrated that all human sGC genes undergo alternative splicing (9, 15, 18, 106, 108). Analysis of sGC mRNA transcripts in the GenBank database reveals a remarkable diversity of human splice isoforms.

NCBI nucleotide database identifies 7 alternatively spliced transcripts for human GUCY1A3 gene of the α1 subunit (α1-Tr1 to α1-Tr7) (64, 107). All α1 transcripts can be sub-divided into two groups based on the encoded protein splice isoforms. The first group includes transcripts 1–4, which encode identical 690 amino acid protein isoform A (canonical α1 sGC) and differ from each other only in their 5′- and 3′-UTR sequences. The second group contains transcripts 5–7, each encoding a different polypeptide named, respectively, isoforms B, C, and D. The domain organization of sGC α1 splice protein variants is presented in Figure 1. These splice variants were characterized biochemically (56, 64, 108).

Based on GeneBank database, in addition to canonical β1 sGC transcript, five more alternative splice isoforms can be identified (64, 107). Domain organization of β1 splice protein variants (Fig. 1) indicates that, with the exception of the canonical β1 subunit (β1-iso2), all alternative splice isoforms of human β1 sGC contain deletions or insertions in the heme-binding domain. Although these β1 splice isoforms are not yet characterized functionally, the location of these insertions and deletions strongly suggest that they will generate sGC with impaired or absent NO-dependent stimulation of cGMP-forming activity.

It appears that the diversity of α1 and β1 sGC splice isoforms is more characteristic for humans. Among mammals, only mice and rats benefit from a sufficiently deep sequencing of the transcriptom to judge with any certainty the diversity of sGC splice forms. As far as the existing RNA-seq data suggest, rats do not have alternative splice transcripts, resulting in an alternatively spliced protein for either subunit (107). There are no predicted splice isoform for the β1 subunit in mice and only one α1 splice transcript that encodes the homolog of the human α1-isoC isoform.

Ritter et al. (94) reported the presence of three alternative α1 sGC splice transcripts in human heart, brain, artery, and B-cells. The translational start site was deleted in these splice transcripts, and no alternative sGC splice protein variants were reported at the time. Nevertheless, it was clear that elevated expression of one of the α1 splice transcripts correlated with decreased sGC activity in immortalized B-lymphocytes.

Later, our laboratories identified several splice variants encoding various polypeptides with deletions in C- and N-termini of sGC (108). We demonstrated that these transcripts are differentially expressed in normal human tissues. Functional characterization of the α1-isoB and α1-isoC splice isoforms revealed that co-expression of these isoforms together with canonical α1/β1 sGC alters the functional properties of sGC. The α1-isoC splice variant, which lost the catalytic domain due to splicing, displayed properties of a dominant negative mutant and inhibited the activity of the α1/β1 sGC heterodimer.

In contrast, the α-isoB, which contains a 240 residue deletion of the N-terminal regulatory domain and an intact catalytic region (Fig. 1), was fully functional and formed an active heterodimer with the β1 subunit, which was robustly activated by NO and allosteric stimulator BAY41-2272. The α1-isoB variant was much less sensitive to degradation induced by ODQ-dependent oxidation in human neuroblastoma cell culture. Under the oxidative pressure from 10 μM ODQ, canonical α1 and β1 splice variants (α1-isoA and β1-iso2) had an estimated half-life of ∼4.3 and 4.6 h, respectively. On the contrary, the α1-isoB splice isoform was stable and its expression increased after cell treatment with ODQ (Fig. 2) (108). Moreover, the expression of this oxidation/degradation-resistant α1-isoB splice isoform stabilized the endogenous β1 subunit and extended its half-life to ∼9.6 h.

FIG. 2.

Co-expression of functional and stable α₁-isoB splice isoform stabilizes β₁ sGC. Time-dependent changes of α₁, α₁-isoB, and β₁ protein quantified by Western blotting in control BE2 cells and BE2 cells expressing α₁-isoB were determined after exposure to 20 μM ODQ, as reported in Sharina et al. (108). The plots are based on the data from Sharina et al. (108) and are shown as mean ± SEM (n = 4 for 6 and 24 h; n = 3 for 12 h). The arrows indicate the shift in the stability of β1 sGC in the presence of α₁-isoB.

The expression of endogenous α1-isoB splice protein was detected in human breast carcinoma MDA468 (20) and, to a lesser extent, in human neuroblastoma BE2 cells (20, 108). Interestingly, the α1-isoB splice isoform was also detected in differentiating human embryonic stem (hES) cells (106). Undifferentiated hES cells do not express sGC (76). However, when hES were allowed to differentiate, the expression of both canonical α1-isoA and α1-isoB splice isoforms increased. In fact, at early stages of differentiation, the 55 kDa α1-isoB constituted more than half of the expressed α1 sGC (106). The expression of this oxidation/degradation-resistant α1-isoB isoform is not restricted just to cell cultures. A 55 kDa α1-isoB immunoreactive band was previously reported in human brain samples (42). Without the benefit of the current bioinformatics data, this 55 kDa band was considered the product of a degradation process that occurs in vivo, but not in vitro. In a recent study, we reported that human aorta also exhibits a high level of 55 kDa α1-isoB protein in human aorta (64).

Besides the transcripts for canonical α1 and β1 subunits and functional α1-isoB variant, a number of transcripts coding for an additional splice variant of both α1 and β1 subunits were detected in normal human aorta (64). It was reported that healthy human aorta expresses mRNA for isoforms C and D of the α1 subunit. The alternative transcripts 3, 5, and 6 for the β1 subunit were also detected. The α1-iso D protein was expressed and characterized biochemically. It was demonstrated that α1-iso D has a significantly impaired capacity for activation by NO or allosteric regulators (64). Biochemical characterization of the α1-isoC splice protein demonstrated that it has a dominant-negative effect on sGC activity due to its lost catalytic region, but preserved capacity to heterodimerize with β1 sGC (108). The proteins coded by the alternative transcripts β1-Tr 3, 5, and 6 are also expected to be dysfunctional. They have missing regions in the catalytic domain or deletions/insertions in the heme-binding region (Fig. 1).

Previous studies revealed that the expression of human α1 sGC splice mRNA may be directly affected by cellular redox conditions. For example, the level of mRNA transcript encoding the canonical α1 sGC (α1-isoA) decreased in MDA468 and BE2 cells exposed to ODQ or H₂O₂. On the contrary, the expression of the mRNA and protein of oxidation/degradation-resistant α1-isoB splice isoform substantially increased in these cellular models (20). This feature is not restricted only to immortalized or transformed cells. As reported here, the treatment with H₂O₂ increased the expression of α1-isoB splice isoform mRNA in the early passage primary human aortic SMCs (Fig. 3).

FIG. 3.

H₂O₂ induces the expression of α₁-Transcript 5 encoding oxidation-resistant α₁-isoB splice variant in healthy VSMC. (A) Early passage human VSMC (PromoCell) were treated with a bolus of 1 mM H₂O₂ and incubated for 24 h, as described in Cote et al. (20). The expression of α1 and α1-isoB sGC and of GAPD transcripts was determined by semi-quantitative RT-PCR. Shown is a representative electrophoretogram out of 3 independent experiments with similar results. (B) Relative expression of α1 and α1-isoB sGC quantified by densitometry and normalized to GAPD mRNA level. H₂O₂, hydrogen peroxide; RT-PCR, reverse transcription polymerase chain reaction; VSMCs, vascular smooth muscle cells.

Increasing evidence points to ROS and RNS as important causal factors in the pathogenesis of aortic aneurysm (70). Aneurysm is a chronic inflammatory condition of the aorta with characteristic structural deficiencies of the vascular wall. In one of our previous studies, we analyzed the composition of canonical and alternative splice variants (64). When commercial quantitative reverse transcription polymerase chain reaction (RT-PCR) probes and primers that recognize regions shared by all α1 or β1 splice variant were used, a 2-3-fold increase in α1 and β1 signals was observed in aortas with aneurysm (64).These data show the same trend as the increased expression of sGC in cholesterol-fed rabbits (57), rats and rabbits with chronic administration of nitroglycerin (79) or elevated expression of β1 sGC in rat aorta at early stages of heart failure (4).

The expression profile of the spliced α1 and β1 transcripts in healthy human aortas and aortas with aneurysm was previously reported (64). In that study, a semi-quantitative RT-PCR analysis based on primers specific for each individual splice form was performed. This approach provided a more accurate picture of the abundance and composition of the splice transcripts in normal aortas and aortas with aneurysm (64). It was determined that aortas with aneurysm have a much higher level of transcripts coding for dysfunctional α1-IsoD and α1-IsoC splice variant. A significant difference between the expression profile for the β1 splice transcripts in normal and diseased aortas was also observed (64). The analysis demonstrated that the level of transcripts that code for dysfunctional isoforms β1-iso1, β1-iso4, and β1-iso6 was also higher in aortas with aneurysm.

The expression of canonical full-length α1 and β1 sGC protein was lower in samples with aneurysm (64) (Fig. 4A). Higher sGC protein degradation in conditions of oxidative stress in aortas with aneurysm is probably the main reason why the level of these proteins did not change, despite the twofold increase in the level of canonical α1 transcript. The shift in the expression profile toward dysfunctional sGC splice forms is probably another factor that contributes to diminished α1 and β1 protein in samples with aneurysm. A recent study of mice expressing only heme-deficient Apo-sGC clearly demonstrated that heme deficiency results in a lower level of sGC protein (125). A similar mechanism probably decreases the expression of sGC in aorta with aneurysm.

FIG. 4.

α1-isoB splice isoform is expressed in normal human aorta, but not in aorta with aneurysm. (A) Six representative samples for normal aorta and aorta with aneurysm were tested by Western blotting by using antibodies against α1 (top panel) or β1 (middle panel) sGC subunits. Recombinant α1 and α1-isoB proteins were used as positive controls (Contr.). Anti-α-actin Western blotting (bottom panel) confirmed equal protein. Western blotting data are from Martin et al. (64). (B) sGC activity is decreased in samples with aneurysm versus control samples. cGMP-forming activities in aortic lysates from control and aneurysm samples in response to 10 μM DEA-NO or 1 μM BAY58-2667 are presented as a percentage of sGC activity in control samples. Circles and squares represent sGC activity determined for individual samples. Means (horizontal bars) ± SD are also shown. NO-stimulated activity of control samples was 100 ± 11 pmole cGMP/25 min. BAY58-2667-stimulated activity of control samples was 36 ± 4 pmole cGMP/25 min. The graphs are calculated based on the data from Martin et al. (64). cGMP, cyclic guanosine monophosphate; DEA-NO, diethylamine-NO.

The measurements of sGC activity in control samples and in samples with aneurysm more directly points to increased content of heme-deficient sGC in aortas with aneurysm. It was reported that in aortas with aneurysm, NO-dependent sGC activity constitutes 64% ± 4% of sGC activity of control aortas (64) (Fig. 4B). This correlates with the lower expression of α1 and β1 sGC and the higher content of dysfunctional sGC. However, the fraction of BAY58-2667-stimuated activity in aortas with aneurysm is higher and constitutes 86% ± 6% of the control (64) (Fig. 4B). The increase in the fraction of BAY58-2667-stimulated sGC supports the notion that samples with aneurysm have an elevated content of sGC with dysfunctional heme.

In the same study, the expression of the oxidation/degradation-resistant α1-IsoB isoform in human aorta was analyzed. The level of α1-IsoB mRNA was lower in samples with aneurysm than in control samples. Correspondingly, the α1-isoB protein was expressed in most normal aortas. In fact, in some samples, this was the major form of the α1 subunit. On the contrary, no signal or only traces of α1-isoB were detected in samples with aneurysm (64) (Fig. 4A). This comparison suggests that α1-isoB may have an important protective function. It is possible that the individuals with higher expression of α1-isoB are less prone to the development of vascular dysfunction, whereas individuals lacking this expression are more susceptible to them.

The exact mechanism behind the changes in sGC splicing under oxidative conditions is not well defined. Initial studies in this area identified a number of splice factors that may be directly responsible for the skipping of the human exon 3 of the α1 gene, which results in the transcript for the α1-isoB protein (20). In silico analysis suggests the existence of several cis-regulatory sequences around the alternative splice site that potentially bind serine/arginine-rich splice factors (SR) or heterogeneous nuclear ribonucleoproteins (hnRNPs) (Fig. 5A). The predicted hnRNP proteins are the polypyrimidine tract-binding protein PTBP1 and the hnRNP A2/B1 isoforms.

FIG. 5.

H₂O₂ selectively alters the expression of splice factors predicted to regulate sGC splicing in MDA468 cells. (A) Binding sites for splicing regulators located in the proximity to the 5′ splice site of α1 sGC exon 4. Constitutive and alternative splice sites for exon 4 of α1 sGC are defined with the UCSC Genome Bioinformatics tool. Their relative consensus values are examined by using Human Splicing Finder. The probability of binding of SR and hnRNP and function as potential regulators of splicing of α1 exon 4 was predicted by Alternative Splicing/Splicing Rainbow tool from European Molecular Biology Laboratory (55). (B) Western blotting for PTB, hnRNP2A/1B, HuR, and SR splicing regulators in lysates from control and H₂O₂-treated (1 mM, 24 h) cells. Representative biological duplicates are shown, and β-actin is included. (C) The level of PTBP1 protein in MDA468 after 24 h treatment with vehicle (phosphate buffered saline [PBS]), 1 mM H₂O₂, 1 mM thiol oxidant HEDS, or 0.01 U/ml of GO, as described in (20). Western blotting in (B) and (C) are reproduction of data from (20). GO, glucose oxidase; hnRNP, heterogeneous nuclear ribonucleoproteins; HuR, human-antigen R. HEDS, bis(2-hydroxyethyl)disulfide.

In a previous report, we demonstrated that the cells exposed overnight to H₂O₂ or cultured in glucose-containing media in the presence of exogenous glucose oxidase (GO) showed no changes in the level of the SR splice factor (20). The level of HuR protein, which stabilizes sGC mRNA, was also not affected. However, a dramatic reduction of PTBP1 and hnRNP A2/B1 proteins was observed (Fig. 5B) (20). A similar H₂O₂-induced reduction was previously reported for closely related hnRNP C1 and D proteins (41).

In another study, the hnRNP A1 homolog was hyperphosphorylated by the stress-induced MKK/p38-pathway, which led to the accumulation of hnRNP A1 in the cytoplasm and affected the splicing in the nucleus (39). A similar crosstalk between hnRNP A2/B1 proteins and p38 kinase is also possible under conditions of oxidative stress, which is a well-established activator of p38 kinase (112). Oxidation of cysteine residues of PTB and hnRNP A2/B1 splice factors is another likely contributor to the splice switching of sGC transcripts. Previous studies demonstrated that the effect of H₂O₂ or GO on PTB in a number of cell cultures was mimicked by bis(2-hydroxyethyl)disulfide (HEDS), a thiol-specific oxidant (20) (Fig. 5). On the other hand, hnRNP A2/B1 was identified as one of the proteins in Hepa 1–6 cells, which contain thiols that are prone to oxidation in response to cell treatments with GO (34).

These pilot studies suggested that PTBP1 and hnRNP A2/B1 may be directly involved in the cellular response, which results in switching to a different set of sGC transcript in response to an acute oxidative episode and/or a chronic low-level oxidative environment that is associated with various pathological conditions (Fig. 6).

FIG. 6.

Role of alternative splicing of sGC transcripts in response to oxidative stress and chronic pathologies. Under the pressure of oxidative stress, the levels of splice factors PTB1 and hnRNP2A/1B decrease, which induces switching to alternative splice forms of sGC. The expression of functional and oxidation-resistant α1-isoB stabilizes sGC and opposes the negative effects of ROS. The positive effects of α1-isoB are counterbalanced by elevated expression of dysfunctional and potentially inhibitory splice forms of α1 and β1 sGC. ROS, reactive oxygen species.

In summary, accumulated evidence allows us to propose that the splicing of sGC is tuned to changes in cellular oxidative condition and plays a dual role in regulation of sGC function (Fig. 6). On the one hand, alternative splicing promotes sGC function via the expression of a stable and functional α1-isoB isoform. On the other hand, the expression of dysfunctional isoforms α1-isoC, α1-isoD, β1-iso1, β1-iso3, β1-iso4, β1-iso5, and β1-iso6 may exacerbate ROS-dependent damage to sGC function and negatively affect sGC stability and activity. Changes in sGC splicing in aortas with aneurysm, where the composition of sGC splice transcripts is altered in favor of transcripts encoding for non-functional or dysfunctional sGC subunit isoforms, is most probably just an example of similar processes taking place in other tissues or under other chronic pathological conditions. These data suggest that sGC splicing is an important, previously unrecognized factor contributing to the development of vascular disease and other pathologies. Oxidative stress persists in diseased blood vessels and increases with age. Increased expression of α1-isoB may be a positive factor contributing to the maintenance of sGC function under oxidative stress conditions. On the contrary, diminished expression of α1-isoB compounded by increased expression of dysfunctional sGC splice forms may be exacerbating the dysfunction of NO/cGMP signaling in diseased tissues.

Future Directions

Targeting the process of sGC splicing in vasculature under oxidative stress may be an important preventive approach to maintain the composition of sGC transcripts expressed in healthy vessels and/or a complementary treatment to existing sGC-targeting therapies. Successful strategies that allow targeted manipulations of RNA splicing are now starting to emerge (136). Modified antisense oligos that are resistant to exonucleases are being developed to manipulate the ratio of expressed transcripts via targeting of alternative RNA splicing. In this approach, the splice-switching oligonucleotides act as competitors for the regulatory sites on the pre-mRNA transcript and sequester the dynamic components of the spliceosome complex responsible for each splicing event. Such targeted competition shifts the splicing process toward the preferred alternative splice site. This approach has been successfully applied to facilitate the expression of a preferred protein isoform and to alter the gene function in a desired manner (8, 100). This strategy was used to achieve exon exclusion, intron retention, exon shuffling, or usage of alternative 5′ and 3′ splice sites (66). Currently, a number of different synthetic oligonucleotides that are resistant to RNase H degradation and high hybridization efficiency and specificity to a specific target were developed (8). The use of modified oligos enabled application of this technique for splicing correction in clinically relevant cultured human cell lines and mouse models (2, 95, 99). Moreover, stable morpholino-oligonucleotides are currently being evaluated in clinical trials for the treatments of β-thalassemia and cystic fibrosis (100). Therefore, understanding the mechanisms that govern the splice switching of sGC transcripts in conditions of oxidative stress or pathological conditions should be beneficial for the development of therapies that modulate sGC function by restoring the composition of sGC splice transcripts featured in healthy tissues under normal conditions.

Footnotes

Acknowledgments

This work was supported by the University of Texas Bridge grant (I.G.S.) and the American Heart Association (grant 15GRNT25700101, E.S.M.). The authors would like to thank Filip Jelen, Elena Bogatenkova, Vladislav Sharin, Eva Golunski, Susan Laing, Anthony Estrera, Gilbert J. Cote, Wen Zhu, Anthony Thomas, Alexander Kots, Kalpana Mujoo, and Ferid Murad for their technical and intellectual contributions to the studies related to sGC splicing.

Abbreviations Used

References

Akaike

, Masuda

, Kuwano

, Nishida

, Kajita

, Kurokawa

, Satake

, Shoda

, Imoto

, and Rokutan

. HuR regulates alternative splicing of the TRA2beta gene in human colon cancer cells under oxidative stress. Mol Cell Biol, 34: 2857–2873, 2014.

Alter

, Lou

, Rabinowitz

, Yin

, Rosenfeld

, Wilton

, Partridge

, and Lu

. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med, 12: 175–177, 2006.

Baltrons

and Garcia

. Nitric oxide-independent down-regulation of soluble guanylyl cyclase by bacterial endotoxin in astroglial cells. J Neurochem, 73: 2149–2157, 1999.

Bauersachs

, Bouloumie

, Fraccarollo

, Hu

, Busse

, and Ertl

. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation, 100: 292–298, 1999.

Bauersachs

, Bouloumie

, Mulsch

, Wiemer

, Fleming

, and Busse

. Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production. Cardiovasc Res, 37: 772–779, 1998.

Bauersachs

, Fleming

, Fraccarollo

, Busse

, and Ertl

. Prevention of endothelial dysfunction in heart failure by vitamin E: attenuation of vascular superoxide anion formation and increase in soluble guanylyl cyclase expression. Cardiovasc Res, 51: 344–350, 2001.

Bauersachs

and Widder

. Endothelial dysfunction in heart failure. Pharmacol Rep, 60: 119–126, 2008.

Bauman

, Jearawiriyapaisarn

, and Kole

. Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides, 19: 1–13, 2009.

Behrends

, Harteneck

, Schultz

, and Koesling

. A variant of the alpha 2 subunit of soluble guanylyl cyclase contains an insert homologous to a region within adenylyl cyclases and functions as a dominant negative protein. J Biol Chem, 270: 21109–21113, 1995.

10.

Brune

, Schmidt

, and Ullrich

. Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion. Eur J Biochem, 192: 683–688, 1990.

11.

Burgoyne

, Madhani

, Cuello

, Charles

, Brennan

, Schroder

, Browning

, and Eaton

. Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science, 317: 1393–1397, 2007.

12.

Burke

and Wolin

. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol, 252: H721–H732, 1987.

13.

Burke-Wolin

and Wolin

. Inhibition of cGMP-associated pulmonary arterial relaxation to H2O2 and O2 by ethanol. Am J Physiol, 258: H1267–H1273, 1990.

14.

Busker

, Neidhardt

, and Behrends

. Nitric oxide activation of guanylate cyclase pushes the alpha1 signaling helix and the beta1 heme-binding domain closer to the substrate-binding site. J Biol Chem, 289: 476–484, 2014.

15.

Cabilla

, Ronchetti

, Nudler

, Miler

, Quinteros

, and Duvilanski

. Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands. Am J Physiol Endocrinol Metab, 296: E731–E737, 2009.

16.

Campbell

, Underbakke

, Potter

, Carragher

, and Marletta

. Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase. Proc Natl Acad Sci U S A, 111: 2960–2965, 2014.

17.

Cherry

and Wolin

. Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase. Free Radic Biol Med, 7: 485–490, 1989.

18.

Chhajlani

, Frandberg

, Ahlner

, Axelsson

, and Wikberg

. Heterogeneity in human soluble guanylate cyclase due to alternative splicing. FEBS Lett, 290: 157–158, 1991.

19.

Costell

, Ancellin

, Bernard

, Zhao

, Upson

, Morgan

, Maniscalco

, Olzinski

, Ballard

, Herry

, Grondin

, Dodic

, Mirguet

, Bouillot

, Gellibert

, Coatney

, Lepore

, Jucker

, Jolivette

, Willette

, Schnackenberg

, and Behm

. Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress. Front Pharmacol, 3: 128, 2012.

20.

Cote

, Zhu

, Thomas

, Martin

, Murad

, and Sharina

. Hydrogen peroxide alters splicing of soluble guanylyl cyclase and selectively modulates expression of splicing regulators in human cancer cells. PLoS One, 7: e41099, 2012.

21.

Crassous

, Couloubaly

, Huang

, Zhou

, Baskaran

, Kim

, Papapetropoulos

, Fioramonti

, Duran

, and Beuve

. Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model. Am J Physiol Heart Circ Physiol, 303: H597–H604, 2012.

22.

Davis

, Martin

, Turko

, and Murad

. Novel effects of nitric oxide. Annu Rev Pharmacol Toxicol, 41: 203–236, 2001.

23.

de Frutos

, Nitta

, Caldwell

, Friedman

, and Gonzalez Bosc

. Regulation of soluble guanylyl cyclase-alpha1 expression in chronic hypoxia-induced pulmonary hypertension: role of NFATc3 and HuR. Am J Physiol Lung Cell Mol Physiol, 297: L475–L486, 2009.

24.

Derbyshire

, Fernhoff

, Deng

, and Marletta

. Nucleotide regulation of soluble guanylate cyclase substrate specificity. Biochemistry, 48: 7519–7524, 2009.

25.

Derbyshire

and Marletta

. Structure and regulation of soluble guanylate cyclase. Annu Rev Biochem, 81: 533–559, 2012.

26.

Dong

, Lu

, Wang

, He

, Chu

, and Ma

. Stabilization of Snail by HuR in the process of hydrogen peroxide induced cell migration. Biochem Biophys Res Commun, 356: 318–321, 2007.

27.

Erusalimsky

and Moncada

. Nitric oxide and mitochondrial signaling: from physiology to pathophysiology. Arterioscler Thromb Vasc Biol, 27: 2524–2531, 2007.

28.

Evgenov

, Pacher

, Schmidt

, Hasko

, Schmidt

, and Stasch

. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential. Nat Rev Drug Discov, 5: 755–768, 2006.

29.

Filippov

, Bloch

, and Bloch

. Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells. J Clin Invest, 100: 942–948, 1997.

30.

Finkel

. Oxidant signals and oxidative stress. Curr Opin Cell Biol, 15: 247–254, 2003.

31.

Francois

and Kojda

. Effect of hypercholesterolemia and of oxidative stress on the nitric oxide-cGMP pathway. Neurochem Int, 45: 955–961, 2004.

32.

Friebe

, Schultz

, and Koesling

. Stimulation of soluble guanylate cyclase by superoxide dismutase is mediated by NO. Biochem J, 335: 527–531, 1998.

33.

Fritz

, Hu

, Brailey

, Berry

, Walker

, and Montfort

. Oxidation and loss of heme in soluble guanylyl cyclase from Manduca sexta. Biochemistry, 50: 5813–5815, 2011.

34.

Fuentes-Almagro

, Prieto-Alamo

, Pueyo

, and Jurado

. Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1–6 cells. J Proteomics, 77: 262–279, 2012.

35.

Garthwaite

. New insight into the functioning of nitric oxide-receptive guanylyl cyclase: physiological and pharmacological implications. Mol Cell Biochem, 334: 221–232, 2010.

36.

Gerassimou

, Kotanidou

, Zhou

, Simoes

, Roussos

, and Papapetropoulos

. Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species. Br J Pharmacol, 150: 1084–1091, 2007.

37.

Ghosh

, Stasch

, Papapetropoulos

, and Stuehr

. Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content. J Biol Chem, 289: 15259–15271, 2014.

38.

Ghosh

and Stuehr

. Soluble guanylyl cyclase requires heat shock protein 90 for heme insertion during maturation of the NO-active enzyme. Proc Natl Acad Sci U S A, 109: 12998–13003, 2012.

39.

Guil

, Long

, and Caceres

. hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol Cell Biol, 26: 5744–5758, 2006.

40.

Hall

and Garthwaite

. What is the real physiological NO concentration in vivo?. Nitric Oxide, 21: 92–103, 2009.

41.

Hayakawa

, Fujikane

, Ito

, Matsumoto

, Nakayama

, and Sekiguchi

. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress. Biochem Biophys Res Commun, 403: 220–224, 2010.

42.

Ibarra

, Nedvetsky

, Gerlach

, Riederer

, and Schmidt

. Regional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain. Brain Res, 907: 54–60, 2001.

43.

Iesaki

, Gupte

, Kaminski

, and Wolin

. Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and H2O2. Am J Physiol, 277: H978–H985, 1999.

44.

Irani

. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res, 87: 179–183, 2000.

45.

Kagota

, Tada

, Nejime

, Nakamura

, Kunitomo

, and Shinozuka

. Chronic production of peroxynitrite in the vascular wall impairs vasorelaxation function in SHR/NDmcr-cp rats, an animal model of metabolic syndrome. J Pharmacol Sci, 109: 556–564, 2009.

46.

Kehrer

. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology, 149: 43–50, 2000.

47.

Kloss

, Bouloumie

, and Mulsch

. Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase. Hypertension, 35: 43–47, 2000.

48.

Kloss

, Furneaux

, and Mulsch

. Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta. J Biol Chem, 278: 2377–2383, 2003.

49.

Kloss

, Rodenbach

, Bordel

, and Mulsch

. Human-antigen R (HuR) expression in hypertension: downregulation of the mRNA stabilizing protein HuR in genetic hypertension. Hypertension, 45: 1200–1206, 2005.

50.

Kloss

, Srivastava

, and Mulsch

. Down-regulation of soluble guanylyl cyclase expression by cyclic AMP is mediated by mRNA-stabilizing protein HuR. Mol Pharmacol, 65: 1440–1451, 2004.

51.

Klug

, Rabani

, and Fridovich

. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J Biol Chem, 247: 4839–4842, 1972.

52.

Kober

, Konig

, Weber

, and Kojda

. Diethyldithiocarbamate inhibits the catalytic activity of xanthine oxidase. FEBS Lett, 551: 99–103, 2003.

53.

Koesling

and Friebe

. Soluble guanylyl cyclase: structure and regulation. Rev Physiol Biochem Pharmacol, 135: 41–65, 1999.

54.

Korkmaz

, Loganathan

, Mikles

, Radovits

, Barnucz

, Hirschberg

, Li

, Hegedus

, Pali

, Weymann

, Karck

, and Szabo

. Nitric oxide- and heme-independent activation of soluble guanylate cyclase attenuates peroxynitrite-induced endothelial dysfunction in rat aorta. J Cardiovasc Pharmacol Ther, 18: 70–77, 2013.

55.

Koscielny

, Le Texier

, Gopalakrishnan

, Kumanduri

, and Riethoven

. ASTD: The Alternative Splicing and Transcript Diversity database. Genomics, 93: 213–220, 2009.

56.

Kraehling

, Busker

, Haase

, Koglin

, Linnenbaum

, and Behrends

. The amino-terminus of nitric oxide sensitive guanylyl cyclase alpha(1) does not affect dimerization but influences subcellular localization. PLoS One, 6: e25772, 2011.

57.

Laber

, Kober

, Schmitz

, Schrammel

, Meyer

, Mayer

, Weber

, and Kojda

. Effect of hypercholesterolemia on expression and function of vascular soluble guanylyl cyclase. Circulation, 105: 855–860, 2002.

58.

Lancaster

Jr.

Nitroxidative, nitrosative, and nitrative stress: kinetic predictions of reactive nitrogen species chemistry under biological conditions. Chem Res Toxicol, 19: 1160–1174, 2006.

59.

, Sayed

, Beuve

, and van den Akker

. NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism. EMBO J, 26: 578–588, 2007.

60.

Maron

, Zhang

, Handy

, Beuve

, Tang

, Loscalzo

, and Leopold

. Aldosterone increases oxidant stress to impair guanylyl cyclase activity by cysteinyl thiol oxidation in vascular smooth muscle cells. J Biol Chem, 284: 7665–7672, 2009.

61.

Marro

, Peiro

, Panayiotou

, Baliga

, Meurer

, Schmidt

, and Hobbs

. Characterization of the human alpha1 beta1 soluble guanylyl cyclase promoter: key role for NF-kappaB(p50) and CCAAT-binding factors in regulating expression of the nitric oxide receptor. J Biol Chem, 283: 20027–20036, 2008.

62.

Martin

, Berka

, Bogatenkova

, Murad

, and Tsai

. Ligand selectivity of soluble guanylyl cyclase: effect of the hydrogen-bonding tyrosine in the distal heme pocket on binding of oxygen, nitric oxide, and carbon monoxide. J Biol Chem, 281: 27836–27845, 2006.

63.

Martin

, Berka

, Sharina

, and Tsai

. Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site. Biochemistry, 51: 2737–2746, 2012.

64.

Martin

, Golunski

, Laing

, Estrera

, and Sharina

. Alternative splicing impairs sGC function in aortic aneurysm. Am J Physiol Heart Circ Physiol, 307: H1565–H1575, 2014.

65.

Martin-Garrido

, Gonzalez-Ramos

, Griera

, Guijarro

, Cannata-Andia

, Rodriguez-Puyol

, and Saura

. H2O2 regulation of vascular function through sGC mRNA stabilization by HuR. Arterioscler Thromb Vasc Biol, 31: 567–573, 2011.

66.

Matlin

, Clark

, and Smith

. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol, 6: 386–398, 2005.

67.

Mayer

, Kleschyov

, Stessel

, Russwurm

, Munzel

, Koesling

, and Schmidt

. Inactivation of soluble guanylate cyclase by stoichiometric S-nitrosation. Mol Pharmacol, 75: 886–891, 2009.

68.

Mayer

, Pfeiffer

, Schrammel

, Koesling

, Schmidt

, and Brunner

. A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione. J Biol Chem, 273: 3264–3270, 1998.

69.

Mayer

, Schrammel

, Klatt

, Koesling

, and Schmidt

. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation. J Biol Chem, 270: 17355–17360, 1995.

70.

McCormick

, Gavrila

, and Weintraub

. Role of oxidative stress in the pathogenesis of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol, 27: 461–469, 2007.

71.

Mergia

, Russwurm

, Zoidl

, and Koesling

. Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain. Cell Signal, 15: 189–195, 2003.

72.

Meurer

, Pioch

, Gross

, and Muller-Esterl

. Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase. J Biol Chem, 280: 33149–33156, 2005.

73.

Meurer

, Pioch

, Pabst

, Opitz

, Schmidt

, Beckhaus

, Wagner

, Matt

, Gegenbauer

, Geschka

, Karas

, Stasch

, Schmidt

, and Muller-Esterl

. Nitric oxide-independent vasodilator rescues heme-oxidized soluble guanylate cyclase from proteasomal degradation. Circ Res, 105: 33–41, 2009.

74.

Mikhed

, Gorlach

, Knaus

, and Daiber

. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair. Redox Biol, 5: 275–289, 2015.

75.

Mittal

and Murad

. Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3′,5′-monophosphate formation. Proc Natl Acad Sci U S A, 74: 4360–4364, 1977.

76.

Mujoo

, Sharin

, Bryan

, Krumenacker

, Sloan

, Parveen

, Nikonoff

, Kots

, and Murad

. Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells. Proc Natl Acad Sci U S A, 105: 18924–18929, 2008.

77.

Mulsch

, Bauersachs

, Schafer

, Stasch

, Kast

, and Busse

. Effect of YC-1, an NO-independent, superoxide-sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators. Br J Pharmacol, 120: 681–689, 1997.

78.

Mulsch

, Luckhoff

, Pohl

, Busse

, and Bassenge

. LY 83583 (6-anilino-5,8-quinolinedione) blocks nitrovasodilator-induced cyclic GMP increases and inhibition of platelet activation. Naunyn Schmiedebergs Arch Pharmacol, 340: 119–125, 1989.

79.

Mulsch

, Oelze

, Kloss

, Mollnau

, Topfer

, Smolenski

, Walter

, Stasch

, Warnholtz

, Hink

, Meinertz

, and Munzel

. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation, 103: 2188–2194, 2001.

80.

Munzel

, Daiber

, and Gori

. Nitrate therapy: new aspects concerning molecular action and tolerance. Circulation, 123: 2132–2144, 2011.

81.

Nalwaya

and Deen

. Nitric oxide, oxygen, and superoxide formation and consumption in macrophage cultures. Chem Res Toxicol, 18: 486–493, 2005.

82.

Napoli

, Paolisso

, Casamassimi

, Al-Omran

, Barbieri

, Sommese

, Infante

, and Ignarro

. Effects of nitric oxide on cell proliferation: novel insights. J Am Coll Cardiol, 62: 89–95, 2013.

83.

Oppermann

, Suvorava

, Freudenberger

, Dao

, Fischer

, Weber

, and Kojda

. Regulation of vascular guanylyl cyclase by endothelial nitric oxide-dependent posttranslational modification. Basic Res Cardiol, 106: 539–549, 2011.

84.

Pacher

, Beckman

, and Liaudet

. Nitric oxide and peroxynitrite in health and disease. Physiol Rev, 87: 315–424, 2007.

85.

Papapetropoulos

, Abou-Mohamed

, Marczin

, Murad

, Caldwell

, and Catravas

. Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta. Br J Pharmacol, 118: 1359–1366, 1996.

86.

Papapetropoulos

, Go

, Murad

, and Catravas

. Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells. Br J Pharmacol, 117: 147–155, 1996.

87.

Papapetropoulos

, Marczin

, Mora

, Milici

, Murad

, and Catravas

. Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. Hypertension, 26: 696–704, 1995.

88.

Papapetropoulos

, Zhou

, Gerassimou

, Yetik

, Venema

, Roussos

, Sessa

, and Catravas

. Interaction between the 90-kDa heat shock protein and soluble guanylyl cyclase: physiological significance and mapping of the domains mediating binding. Mol Pharmacol, 68: 1133–1141, 2005.

89.

Parker

and Gori

. Tolerance to the organic nitrates: new ideas, new mechanisms, continued mystery. Circulation, 104: 2263–2265, 2001.

90.

Pedraza

, Baltrons

, Heneka

, and Garcia

. Interleukin-1 beta and lipopolysaccharide decrease soluble guanylyl cyclase in brain cells: NO-independent destabilization of protein and NO-dependent decrease of mRNA. J Neuroimmunol, 144: 80–90, 2003.

91.

Pick

, Rabani

, Yost

, and Fridovich

. The catalytic mechanism of the manganese-containing superoxide dismutase of Escherichia coli studied by pulse radiolysis. J Am Chem Soc, 96: 7329–7333, 1974.

92.

Priviero

, Zemse

, Teixeira

, and Webb

. Oxidative stress impairs vasorelaxation induced by the soluble guanylyl cyclase activator BAY 41-2272 in spontaneously hypertensive rats. Am J Hypertens, 22: 493–499, 2009.

93.

Quijano

, Alvarez

, Gatti

, Augusto

, and Radi

. Pathways of peroxynitrite oxidation of thiol groups. Biochem J, 322: 167–173, 1997.

94.

Ritter

, Taylor

, Hoffmann

, Carnaghi

, Giddings

, Zakeri

, and Kwok

. Alternative splicing for the alpha1 subunit of soluble guanylate cyclase. Biochem J, 346 Pt 3: 811–816, 2000.

95.

Roberts

, Palma

, Sazani

, Orum

, Cho

, and Kole

. Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice. Mol Ther, 14: 471–475, 2006.

96.

Ruetten

, Zabel

, Linz

, and Schmidt

. Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats. Circ Res, 85: 534–541, 1999.

97.

Sayed

, Baskaran

, Ma

, van den Akker

, and Beuve

. Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation. Proc Natl Acad Sci U S A, 104: 12312–12317, 2007.

98.

Sayed

, Kim

, Fioramonti

, Iwahashi

, Duran

, and Beuve

. Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance. Circ Res, 103: 606–614, 2008.

99.

Sazani

, Gemignani

, Kang

, Maier

, Manoharan

, Persmark

, Bortner

, and Kole

. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol, 20: 1228–1233, 2002.

100.

Sazani

and Kole

. Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J Clin Invest, 112: 481–486, 2003.

101.

Schindler

, Strobel

, Schonafinger

, Linz

, Lohn

, Martorana

, Rutten

, Schindler

, Busch

, Sohn

, Topfer

, Pistorius

, Jannek

, and Mulsch

. Biochemistry and pharmacology of novel anthranilic acid derivatives activating heme-oxidized soluble guanylyl cyclase. Mol Pharmacol, 69: 1260–1268, 2006.

102.

Schmidt

, Schmidt

, and Stasch

. NO- and haem-independent soluble guanylate cyclase activators. Handb Exp Pharmacol (191): 309–339, 2009.

103.

Scott

and Nakayama

. Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle. J Surg Res, 80: 309–314, 1998.

104.

Scott

and Nakayama

. Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle. J Surg Res, 79: 66–70, 1998.

105.

Seeger

, Quintyn

, Tanimoto

, Williams

, Tainer

, Wysocki

, and Garcin

. Interfacial residues promote an optimal alignment of the catalytic center in human soluble guanylate cyclase: heterodimerization is required but not sufficient for activity. Biochemistry, 53: 2153–2165, 2014.

106.

Sharin

, Mujoo

, Kots

, Martin

, Murad

, and Sharina

. Nitric oxide receptor soluble guanylyl cyclase undergoes splicing regulation in differentiating human embryonic cells. Stem Cells Dev, 20: 1287–1293, 2010.

107.

Sharina

, Cote

, Martin

, Doursout

, and Murad

. RNA splicing in regulation of nitric oxide receptor soluble guanylyl cyclase. Nitric Oxide, 25: 265–274, 2011.

108.

Sharina

, Jelen

, Bogatenkova

, Thomas

, Martin

, and Murad

. Alpha1 soluble guanylyl cyclase (sGC) splice forms as potential regulators of human sGC activity. J Biol Chem, 283: 15104–15113, 2008.

109.

Sharina

, Krumenacker

, Martin

, and Murad

. Genomic organization of alpha1 and beta1 subunits of the mammalian soluble guanylyl cyclase genes. Proc Natl Acad Sci U S A, 97: 10878–10883, 2000.

110.

Sharina

, Martin

, Thomas

, Uray

, and Murad

. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line. Proc Natl Acad Sci U S A, 100: 11523–11528, 2003.

111.

Silva

, Lanaro

, Leiria

, Rodrigues

, Davel

, Claudino

, Toque

, and Antunes

. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum. Am J Physiol Heart Circ Physiol, 307: H1393–H1400, 2014.

112.

Son

, Kim

, Chung

, and Pae

. Reactive oxygen species in the activation of MAP kinases. Methods Enzymol, 528: 27–48, 2013.

113.

Stasch

and Hobbs

. NO-independent, haem-dependent soluble guanylate cyclase stimulators. Handb Exp Pharmacol (191): 277–308, 2009.

114.

Stasch

, Pacher

, and Evgenov

. Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease. Circulation, 123: 2263–2273, 2011.

115.

Stasch

, Schmidt

, Alonso-Alija

, Apeler

, Dembowsky

, Haerter

, Heil

, Minuth

, Perzborn

, Pleiss

, Schramm

, Schroeder

, Schroder

, Stahl

, Steinke

, and Wunder

. NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle. Br J Pharmacol, 136: 773–783, 2002.

116.

Stasch

, Schmidt

, Nedvetsky

, Nedvetskaya

, H S AK, Meurer

, Deile

, Taye

, Knorr

, Lapp

, Muller

, Turgay

, Rothkegel

, Tersteegen

, Kemp-Harper

, Muller-Esterl

, and Schmidt

. Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels. J Clin Invest, 116: 2552–2561, 2006.

117.

Stone

. An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems. Arch Biochem Biophys, 422: 119–124, 2004.

118.

, Mitra

, Gregg

, Flavahan

, Chotani

, Clark

, Goldschmidt-Clermont

, and Flavahan

. Redox regulation of vascular smooth muscle cell differentiation. Circ Res, 89: 39–46, 2001.

119.

Suliman

and Piantadosi

. Mitochondrial biogenesis: regulation by endogenous gases during inflammation and organ stress. Curr Pharm Des, 20: 5653–5662, 2014.

120.

Takata

, Filippov

, Liu

, Ichinose

, Janssens

, Bloch

, and Bloch

. Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms. Am J Physiol Lung Cell Mol Physiol, 280: L272–L278, 2001.

121.

Tawa

, Geddawy

, Shimosato

, Iwasaki

, Imamura

, and Okamura

. Soluble guanylate cyclase redox state under hypoxia or hypoxia/reoxygenation in isolated monkey coronary arteries. J Pharmacol Sci, 125: 169–175, 2014.

122.

Tawa

, Shimosato

, Iwasaki

, Imamura

, and Okamura

. Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries. Free Radic Res, 49: 1–18, 2015.

123.

Tawa

, Shimosato

, Iwasaki

, Imamura

, and Okamura

. Effects of peroxynitrite on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries. J Vasc Res, 51: 439–446, 2015.

124.

Thomas

, Schulz

, and Keaney

Jr . Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity—role for iron-dependent oxidative stress. Free Radic Biol Med, 41: 681–688, 2006.

125.

Thoonen

, Cauwels

, Decaluwe

, Geschka

, Tainsh

, Delanghe

, Hochepied

, De Cauwer

, Rogge

, Voet

, Sips

, Karas

, Bloch

, Vuylsteke

, Stasch

, Van de Voorde

, Buys

, and Brouckaert

. Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice. Nat Commun, 6: 8482, 2015.

126.

Tsai

, Berka

, Martin

, and Olson

. A “sliding scale rule” for selectivity among NO, CO, and O(2) by heme protein sensors. Biochemistry, 51: 172–186, 2011.

127.

Tsai

, Berka

, Sharina

, and Martin

. Dynamic ligand exchange in soluble guanylyl cyclase (sGC): implications for sGC regulation and desensitization. J Biol Chem, 286: 43182–43192, 2011.

128.

Tsai

, Martin

, Berka

, and Olson

. How do heme-protein sensors exclude oxygen? Lessons learned from cytochrome c′, Nostoc puntiforme heme nitric oxide/oxygen-binding domain, and soluble guanylyl cyclase. Antioxid Redox Signal, 17: 1246–1263, 2012.

129.

Ujiie

, Hogarth

, Danziger

, Drewett

, Yuen

, Pang

, and Star

. Homologous and heterologous desensitization of a guanylyl cyclase-linked nitric oxide receptor in cultured rat medullary interstitial cells. J Pharmacol Exp Ther, 270: 761–767, 1994.

130.

Underbakke

, Iavarone

, Chalmers

, Pascal

, Novick

, Griffin

, and Marletta

. Nitric oxide-induced conformational changes in soluble guanylate cyclase. Structure, 22: 602–611, 2014.

131.

Waldman

, Rapoport

, Ginsburg

, and Murad

. Desensitization to nitroglycerin in vascular smooth muscle from rat and human. Biochem Pharmacol, 35: 3525–3531, 1986.

132.

Wang

, Sandberg

, Luo

, Khrebtukova

, Zhang

, Mayr

, Kingsmore

, Schroth

, and Burge

. Alternative isoform regulation in human tissue transcriptomes. Nature, 456: 470–476, 2008.

133.

Weber

, Lauer

, Mulsch

, and Kojda

. The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase. Free Radic Biol Med, 31: 1360–1367, 2001.

134.

Wedgwood

, Steinhorn

, Bunderson

, Wilham

, Lakshminrusimha

, Brennan

, and Black

. Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn. Am J Physiol Lung Cell Mol Physiol, 289: L660–L666, 2005.

135.

White

, Crawford

, Patt

, and Lad

. Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide. J Biol Chem, 251: 7304–7312, 1976.

136.

Wilton

and Fletcher

. RNA splicing manipulation: strategies to modify gene expression for a variety of therapeutic outcomes. Curr Gene Ther, 11: 259–275, 2011.

137.

Winterbourn

. Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol, 4: 278–286, 2008.

138.

, Pritchard

Jr. , Kaminski

, Fayngersh

, Hintze

, and Wolin

. Involvement of nitric oxide and nitrosothiols in relaxation of pulmonary arteries to peroxynitrite. Am J Physiol, 266: H2108–H2113, 1994.

139.

Zhao

, Brandish

, Ballou

, and Marletta

. A molecular basis for nitric oxide sensing by soluble guanylate cyclase. Proc Natl Acad Sci U S A, 96: 14753–14758, 1999.

140.

Zhou

, Pyriochou

, Kotanidou

, Dalkas

, van Eickels

, Spyroulias

, Roussos

, and Papapetropoulos

. Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress. Am J Physiol Heart Circ Physiol, 295: H1763–H1771, 2008.

The Role of Reactive Oxygen and Nitrogen Species in the Expression and Splicing of Nitric Oxide Receptor

Abstract

Significance:

Recent Advances:

Critical Issues:

Future Directions:

Introduction

ROS/RNS and sGC

Effects of O2 −

Effects of ONOO−

Effect of sGC nitrosation

Effect of H2O2

Effect of complex oxidative stress on sGC function

Effect of ROS/RNS on sGC expression

sGC Splice Variants and Oxidative Stress

Future Directions

Footnotes

Acknowledgments

Abbreviations Used

References

Effects of O₂ ⁻

Effects of ONOO⁻

Effect of H₂O₂