Downregulation of the α1- and ß1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent.

The nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and ß subunit. NO binds to heme of the ß1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1ß1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)-positive and HPV-negative OPSCC contributes to the regulation of sGCα1ß1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα1ß1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α1- and ß1-subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α1- and ß1-subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the ß1-subunit, and thus, α1- and ß1-subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα1ß1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα1ß1 in arterial VSMCs.

Nitrergic signaling via interstitial cells of Cajal and smooth muscle cells influences circular smooth muscle contractility in murine colon.

BACKGROUND: Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO-sensitive guanylyl cyclase (NO-GC). In the GI tract, NO-GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell-specific knockout mice, we have previously shown that NO-GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear. METHODS: Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5 mm) from global (GCKO) and cell-specific knockout mice for NO-GC. Immunohistochemistry and in situ hybridization were used to specify NO-GC expression. KEY RESULTS: Colonic circular smooth muscle showed three different contraction patterns: high-frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO-GC. Slow phasic contractions occurred intermittently in WT, SMC-GCKO, and ICC-GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC-GCKO tissue. Tetrodotoxin and the NO-GC inhibitor ODQ transformed slow phasic contractions of WT and single cell-specific knockout into GCKO-like contractions. ODQ increased the frequency of large contractions in WT and ICC-GCKO colon but not in GCKO, SMC-GCKO, and SMC/ICC-GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions. CONCLUSIONS AND INFERENCES: We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO-GC in SMC determines smooth muscle tone, concerted action of NO-GC in both SMC and ICC modulates slow phasic contractions and large contractions.

Comparison of nitrergic signaling in circular and longitudinal smooth muscle of murine ileum.

BACKGROUND: Gastrointestinal (GI) motility originates from coordinated movements of circular (CM) and longitudinal (LM) smooth muscle. How the two muscle layers react individually to nitrergic input and how they integrate nitrergic signaling is not thoroughly understood. METHODS: We used immunohistochemistry to unveil expression of NO-sensitive guanylyl cyclase (NO-GC) in the ileum. For functional analyses, we measured tone of ileal CM and spontaneous contractions in both ileal muscle layers from mice lacking NO-GC globally (GCKO) and specifically in smooth muscle cells (SMC-GCKO). KEY RESULTS: In contrast to other parts of the GI tract, NO-GC was not expressed in ckit-positive cells in ileum. NO-GC expression was intense in platelet-derived growth factor receptor α-positive cells and in yet unidentified cells of myenteric plexus and serosa. Both CM and LM developed spontaneous contractile activity; frequency and duration of their spontaneous contractions were identical. The amplitude of spontaneous contractions in CM was increased in the absence of NO-GC. In ileum from control (ctrl) animals, inhibition of NO-GC increased whereas NO-GC stimulation decreased tissue tone. In contrast, contractile activity in LM was not different between ctrl and knockout strains. Here, NO led to suppression of spontaneous contractions of ctrl ileum whereas GCKO tissue was unaffected. To our surprise, NO suppressed spontaneous contractions in SMC-GCKO ileum indicating participation of other cell type(s). CONCLUSIONS AND INFERENCES: NO-GC in SMC is involved in the regulation of tone and amplitude of spontaneous contractions in ileal CM. In LM, NO induces suppression of spontaneous contractions via NO-GC in a non-SMC type.

Drivers of hibernation in the brown bear.

BACKGROUND: Hibernation has been a key area of research for several decades, essentially in small mammals in the laboratory, yet we know very little about what triggers or ends it in the wild. Do climatic factors, an internal biological clock, or physiological processes dominate? Using state-of-the-art tracking and monitoring technology on fourteen free-ranging brown bears over three winters, we recorded movement, heart rate (HR), heart rate variability (HRV), body temperature (Tb), physical activity, ambient temperature (TA), and snow depth to identify the drivers of the start and end of hibernation. We used behavioral change point analyses to estimate the start and end of hibernation and convergent cross mapping to identify the causal interactions between the ecological and physiological variables over time. RESULTS: To our knowledge, we have built the first chronology of both ecological and physiological events from before the start to the end of hibernation in the field. Activity, HR, and Tb started to drop slowly several weeks before den entry. Bears entered the den when snow arrived and when ambient temperature reached 0 °C. HRV, taken as a proxy of sympathetic nervous system activity, dropped dramatically once the bear entered the den. This indirectly suggests that denning is tightly coupled to metabolic suppression. During arousal, the unexpected early rise in Tb (two months before den exit) was driven by TA, but was independent of HRV. The difference between Tb and TA decreased gradually suggesting that bears were not thermoconforming. HRV increased only three weeks before exit, indicating that late activation of the sympathetic nervous system likely finalized restoration of euthermic metabolism. Interestingly, it was not until TA reached the presumed lower critical temperature, likely indicating that the bears were seeking thermoneutrality, that they exited the den. CONCLUSIONS: We conclude that brown bear hibernation was initiated primarily by environmental cues, but terminated by physiological cues.

The sGC stimulator riociguat inhibits platelet function in washed platelets but not in whole blood.

BACKGROUND AND PURPOSE: Stimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. EXPERIMENTAL APPROACH: The effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. KEY RESULTS: Riociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50-100 µM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. CONCLUSIONS AND IMPLICATIONS: Riociguat inhibits platelet activation in whole blood only at concentrations above 50 µM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered.

Toward a better understanding of gastrointestinal nitrergic neuromuscular transmission.

BACKGROUND: Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal (GI) tract. The majority of nitrergic effects are transduced by NO-sensitive guanylyl cyclase (NO-GC) as the receptor for NO, and, thus, mediated by cGMP-dependent mechanisms. Work carried out during the past years has demonstrated NO to be largely involved in GI smooth muscle relaxation and motility. However, detailed investigation of nitrergic signaling has turned out to be complicated as NO-GC was identified in several different GI cell types such as smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. With regards to nitrergic neurotransmission, special focus has been placed on the role of interstitial cells of Cajal using mutant mice with reduced populations of ICC. Recently, global and cell-specific knockout mice for enzymes participating in nitrergic signaling have been generated providing a suitable approach to further examine the role of NO-mediated signaling in GI smooth muscle. PURPOSE: This review discusses the current knowledge on nitrergic mechanisms in gastrointestinal neuromuscular transmission with a focus on genetic models and outlines possible further investigations to gain better understanding on NO-mediated effects in the GI tract.

Therapeutic immunomodulation using a virus--the potential of inactivated orf virus.

Viruses can manipulate the immune response against them by various strategies to influence immune cells, i.e. by over-activation leading to functional inactivation, bypassing antigen presentation or even suppression of effector functions. Little is known, however, about how these features of immune regulation and modulation could be used for therapeutic purposes. Reasons for this include the complexity of immune regulatory mechanisms under certain disease conditions and the risks that infections with viruses pose to human beings. The orf virus (ORFV), a member of the Parapoxvirus genus of the poxvirus family, is known as a common pathogen in sheep and goats worldwide. The inactivated ORFV, however, has been used as a preventative as well as therapeutic immunomodulator in veterinary medicine in different species. Here, we review the key results obtained in pre-clinical studies or clinical studies in veterinary medicine to characterise the therapeutic potential of inactivated ORFV. Inactivated ORFV has strong effects on cytokine secretion in mice and human immune cells, leading to an auto-regulated loop of initial up-regulation of inflammatory and Th1-related cytokines, followed by Th2-related cytokines that attenuate immunopathology. The therapeutic potential of inactivated ORFV has been recognised in several difficult-to-treat disease areas, such as chronic viral diseases, liver fibrosis or various forms of cancer. Further research will be required in order to evaluate the full beneficial potential of inactivated ORFV for therapeutic immunomodulation.

Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase.

Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

Stress increases VCAM-1 expression at the fetomaternal interface in an abortion-prone mouse model.

Sound stress exposure increases fetal loss via inflammatory pathways. Inflammation is known to up-regulate cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), which mediates the adhesion of leukocytes to the vascular endothelium. In this work, we studied the frequency of VCAM-1(+) vessels at the fetomaternal interface in stressed and non-stressed pregnant CBA/J female mice mated with DBA/2J (high fetal loss model) or BALB/c (low fetal loss model) males. The high fetal loss model had fewer large vessels on gestation day 6.5, and stress reduced the frequency of large vessels to a similar number in both high and low fetal loss models. In the high fetal loss model, however, the frequency of VCAM-1+ vessels was dramatically increased. This study shows that VCAM-1 expression is modulated by stress at the fetomaternal interface in abortion-prone cross-breeding.

Nitric oxide-sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition.

BACKGROUND: The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade is involved in the precise regulation of platelet responses. NO released from the endothelium is known to activate NO-sensitive guanylyl cyclase (NO-GC) in platelets. By the generation of cGMP and subsequent activation of cGMP-dependent protein kinase (PKG), NO-GC mediates the reduction of the intracellular calcium and inhibits platelet adhesion and aggregation. However, NO has been postulated to influence these platelet functions also via cGMP-independent mechanisms. OBJECTIVE: We studied the effect of NO on platelets lacking NO-sensitive guanylyl cyclase with regards to aggregation, adhesion, calcium mobilization and bleeding time. METHODS AND RESULTS: Here, we show that NO signaling leading to inhibition of agonist-induced platelet aggregation is totally abrogated in platelets from mice deficient in NO-GC (GCKO). Even at millimolar concentrations none of the several different NO donors inhibited collagen-induced aggregation of GCKO platelets. In addition, NO neither affected adenosine 5'-diphosphate (ADP)-induced adhesion nor thrombin-induced calcium release in GCKO platelets. Although the NO-induced cGMP signal transduction was totally abrogated cyclic adenosine monophosphate (cAMP) signaling was still functional; however, cGMP/cAMP crosstalk was disturbed on the level of phosphodiesterase type 3 (PDE3). These in vitro data are completed by a reduced bleeding time indicating the lack of NO effect in vivo. CONCLUSIONS: We conclude that NO-GC is the only NO receptor in murine platelets mediating the inhibition of calcium release, adhesion and aggregation: lack of the enzyme leads to disturbance of primary hemostasis.

Negative feedback in NO/cGMP signalling.

Most of the effects of the signalling molecule nitric oxide (NO) are mediated by the stimulation of the NO-sensitive GC (guanylate cyclase) and the subsequent increase in cGMP formation. The enzyme contains a prosthetic haem group, which mediates NO stimulation. In addition to the physiological activator NO, NO-sensitizers like the substance YC-1 sensitize the enzyme towards NO and may therefore have important pharmacological implications. Two isoforms of NO-sensitive GC have been identified to date that share regulatory properties, but differ in the subcellular localization. The more ubiquitously expressed alpha1beta1 heterodimer and the alpha2beta1 isoform are mainly expressed in brain. In intact cells, NO-induced cGMP signalling not only depends on cGMP formation, but is also critically determined by the activity of the enzymes responsible for cGMP degradation, e.g. PDE5 (phosphodiesterase 5). Recently, direct activation of PDE5 by cGMP was demonstrated, limiting the cGMP increase and thus functioning as a negative feedback. As the cGMP-induced PDE5 activation turned out to be sustained, in the range of hours, it is probably responsible for the NO-induced desensitization observed within NO/cGMP signalling.

Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme.

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

Synergism between nitric oxide and hydrogen peroxide in the inhibition of platelet function: the roles of soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein.

In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition.

Acute blood pressure effects of YC-1-induced activation of soluble guanylyl cyclase in normotensive and hypertensive rats.

We used YC-1 as a pharmacological tool to investigate the short-term blood pressure effects of NO-independent activation of sGC in normotensive and hypertensive rats. Four groups of normotensive Wistar-Kyoto rats were treated by i.v. injection with vehicle (V), YC-1 (YC-1), sodium nitroprusside (SNP), or YC-1 and SNP (YC-1+SNP). Hypertension was induced in four additional groups of WKY rats by 3 weeks of oral treatment with L-NAME. These animals were investigated with the same protocol as the normotensive animals: L-NAME/V, L-NAME/YC-1, L-NAME/SNP, L-NAME/YC-1+SNP. YC-1 lowered mean arterial blood pressure (MAP) in normotensive and hypertensive animals similarly to SNP alone (P<0.05, respectively). The combination of YC-1 with SNP caused a strong decrease of MAP in both the hypertensive and normotensive animals (P<0.05, respectively). SNP with YC-1 also induced a pronounced cyclic GMP increase in the aorta. This study shows for the first time the blood pressure lowering potential of bimodal targeting of the NO-sGC-system.

A behavioral test of presbycusis in the bird auditory system.

Absolute auditory thresholds were determined behaviorally in European starlings (Sturnus vulgaris) between the age of 6 months and up to 13 years using a GO/NOGO procedure. The thresholds that we observed in individual starlings over a time period of 11 years showed no systematic increase over time. When comparing young starlings (age 6 to 12 months) with old starlings (age 8 to 13 years), we discovered no substantial age-related hearing loss. In the frequency range from 0.5 to 4 kHz, the thresholds of old subjects were on average increased by 1.5 to 3 dB. For frequencies of 6 and 8 kHz, the mean threshold increase of old subjects was 6.1 and 4.9 dB, respectively. This demonstrates excellent hearing in subjects that had lived on average more than five times the starlings' demographic life span of 22 months. This result is discussed with respect to the large threshold shift usually found in aging mammals and to differences between the bird and the mammalian auditory system.

A point-mutated guanylyl cyclase with features of the YC-1-stimulated enzyme: implications for the YC-1 binding site?

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in various signaling cascades and are structurally closely related. The crystal structure of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the heterodimeric soluble GC (sGC), has been identified which acts like forskolin on AC. Here, we investigated the respective substrate and potential activator domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost complete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and higher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain which corresponds to the forskolin site of the ACs may comprise the binding site for YC-1.

15-Lipoxygenase catalytically consumes nitric oxide and impairs activation of guanylate cyclase.

Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (.NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of .NO (up to 2 microM) with either native (Ered) or ferric LOXs (0.2 microM) metal centers to form nitrosyl complexes occurred at these .NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of .NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that.NO (2.2 microM) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, .NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of .NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by .NO is proposed whereby reaction of .NO with EredLOO. generates Ered and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2-). Reactivation of Ered, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of .NO with 15-LOX inhibited. NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated .NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of .NO with lipoxygenase peroxyl radical intermediates will result in modulation of both .NO bioavailability and rates of production of lipid signaling mediators.

YC-1 potentiates nitric oxide- and carbon monoxide-induced cyclic GMP effects in human platelets.

Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.

Stimulation of soluble guanylate cyclase by superoxide dismutase is mediated by NO.

Soluble guanylate cyclase (sGC), which is found in many cells and tissues, represents the receptor for the intra- and intercellular messenger molecule NO. Superoxide dismutase (SOD), an enzyme involved in the degradation of toxic superoxide radicals, has been proposed as a non-NO activator of sGC. Here we show that SOD stimulated sGC purified from bovine lung up to 10-fold. Activation by SOD was not influenced by the hydroxyl radical scavengers mannitol and DMSO. In contrast, the presence of the NO scavengers oxyhaemoglobin and 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide, as well as the O2(-)-generating system xanthine oxidase/hypoxanthine, led to inhibition of SOD-stimulated cGMP production. NO-insensitive sGC mutants were not influenced either by SOD or by xanthine oxidase. We have previously shown that sGC was stimulated by NO present in the normal atmosphere. Here we show that the SOD effect depended on the NO concentration from the atmosphere, as the stimulation of sGC by defined NO gases (0, 120, 330 and 1000 parts per billion NO) was potentiated by SOD. NO stimulation of sGC and its potentiation by SOD were inhibited by oxyhaemoglobin to identical levels. We conclude that the SOD-mediated stimulation of sGC is due to the elimination of superoxide, thereby preventing its reaction with NO to form peroxynitrite.