Deconstructing endothelial dysfunction: soluble guanylyl cyclase oxidation and the NO resistance syndrome

In this issue of the JCI, Stasch and colleagues suggest that a novel drug, BAY 58-2667, potently activates a pool of oxidized and heme-free soluble guanylyl cyclase (sGC; see the related article beginning on page 2552). The increased vasodilatory potency of BAY 58-2667 the authors found in a number of animal models of endothelial dysfunction and in human blood vessels from patients with diabetes suggests that there exists a subphenotype of endothelial dysfunction characterized by receptor-level NO resistance. Diseases associated with NO resistance would appear to be ideally suited for therapies directed at restoring redox homeostasis, sGC activity, and NO sensitivity.     

Soluble guanylyl cyclase: physiological role as an NO receptor and the potential molecular target for therapeutic application.

Nitric oxide (NO) activates soluble guanylyl cyclase, which results in an increased synthesis of cyclic guanosine 3',5'-cyclic monophosphate (cGMP), smooth muscle relaxation and vasodilation. The heme group in soluble guanylyl cyclase binds NO and allosterically activates the catalytic site. In addition, a second allosteric site that synergistically activates the enzyme has been reported. BAY 41-2272 was reported as an NO-independent activator of soluble guanylyl cyclase. Treatment with this compound results in anti-platelet activity, a decrease in blood pressure and an increase in survival, indicating a potential for treating cardiovascular diseases. YC-1, another NO-independent activator, activates soluble guanylyl cyclase and the activity is enhanced in the presence of NO. YC-1 relaxed tissue strips in organ bath. Consistent with its biochemical activity, YC-1 induced penile erection in a conscious rat model. Recently, we found a novel series of soluble guanylyl cyclase activators that also NO-independently activate soluble guanylyl cyclase and cause penile erection, suggesting a synergy with the endogenous NO production in vivo. Here I review the NO/cGMP signal transduction pathway and define soluble guanylyl cyclase modulators as a novel approach for the treatment of cardiovascular diseases and erectile dysfunction.     

Inhibitory phosphorylation of soluble guanylyl cyclase by muscarinic m2 receptors via Gbetagamma-dependent activation of c-Src kinase

In gastrointestinal smooth muscle, cGMP levels in response to relaxant agonists are regulated by activation of phosphodiesterase 5 and inhibition of soluble guanylyl cyclase (sGC) in a feedback mechanism via cGMP-dependent protein kinase. The aim of the present study was to determine whether contractile agonists modulate cGMP levels by cross-regulating sGC activity. In gastric muscle cells, acetylcholine (ACh) stimulated Src activity and induced sGC phosphorylation. Concurrent stimulation of cells with ACh attenuated sGC activity and cGMP formation in response to the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). The effect of ACh on Src activity, sGC phosphorylation, and on GSNO-stimulated sGC activity and cGMP formation were blocked by the m2 receptor antagonist (methoctramine), pertussis toxin, and by inhibitors of phosphatidylinositol 3 kinase, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or Src kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, in dispersed muscle cells and in cells expressing Galpha(i) minigene or Gbetagamma-scavenging peptide, whereas the m3 receptor antagonist, N-(2-chloroethyl)-4-piperidinyl diphenylacetate, or expression of the Galpha(q) minigene had no effect. ACh also attenuated sGC activity and cGMP formation in response to the NO-independent activator, YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzylindazole]. The pattern implied that phosphorylation of sGC by c-Src kinase inhibits NO-sensitive sGC activity, and the inhibition was not due to a decrease in the binding of NO but probably due to decrease in catalytic activity. We conclude that cGMP levels are cross-regulated by contractile agonists via a mechanism that involves c-Src-dependent phosphorylation of sGC, leading to inhibition of sGC activity and cGMP formation. The finding highlights a novel mechanism for attenuation of the NO/sGC/cGMP signal by G(i)-coupled contractile agonists, in addition to their inhibitory effect on adenylyl cyclase and cAMP formation.     

鸟苷酸环化酶C在大肠癌患者外周血中的表达及其临床意义

目的：探讨鸟甘酸环化酶 C （ GCC ）在大肠癌患者外周血中的表达及其临床意义。方法采用RT-PCR法对32例大肠癌患者、25例大肠良性病变患者、30例健康体检者外周血中GCC表达情况进行检测,并对大肠癌病理指标与GCC mRNA 表达阳性率之间关系进行研究。结果32例大肠癌患者中,GCC mRNA 的阳性检出率为65.63％（21／32）,良性组中 GCC mRNA 的阳性检出率为20.00％（5／25）,而对照组中未见 GCC mRNA 表达;三组外周血中表达阳性率差异具有统计学意义（P〈0.05）。大肠癌患者 GCC mRNA 表达水平与Duke 分期、淋巴结转移和肝转移密切相关（P〈0.05）,与年龄、性别、肿瘤大小等无关（P〉0.05）。结论 GCC mRNA 在大肠癌患者外周血中阳性表达率较高,特别是复发或转移病例,可作为大肠癌术后复发、转移的早期检测指标之一。     

Hydroxyurea induces fetal hemoglobin by the nitric oxide-dependent activation of soluble guanylyl cyclase

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of gamma-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced gamma-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.     

Effects of a novel guanylyl cyclase inhibitor on the vascular actions of nitric oxide and peroxynitrite in immunostimulated smooth muscle cells and in endotoxic shock.

OBJECTIVE: Nitric oxide (NO), produced by the inducible isoform of NO synthase (NOS) in circulatory shock exerts cytotoxic and vasodilator effects. Part of these effects are mediated by formation of peroxynitrite, a toxic oxidant produced by the rapid reaction of NO and superoxide. Other parts of the vascular actions of NO in shock are thought to be mediated by the action of NO on the soluble guanylyl cyclase (GC) in the smooth muscle and subsequent decrease in the intracellular calcium levels. Using 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1 -one (ODQ), a potent inhibitor of GC, we studied the role of GC activation in the NO- and peroxynitrite-related vascular alterations. DESIGN: In vitro: Controlled experiment using cultured rat aortic smooth muscle cells. In vivo: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Male Wistar rats and male Swiss mice. Interventions: In vitro: a) Stimulation of rat aortic smooth muscle cells with bacterial lipopolysaccharide (LPS) and gamma-interferon, measurement of the production of nitrite and nitrate (breakdown products of NO), and suppression of mitochondrial respiration for 24 to 48 hrs, in the presence or absence of ODQ; and b) in norepinephrine-precontracted endothelium-denuded thoracic aortic rings, exposure to LPS (10 ng/mL) in the presence or absence of ODQ. In vivo: Rats treated in vivo with LPS (10 mg/kg iv for 3 hrs) and mice challenged with 60 mg/kg LPS ip, in the presence or absence of ODQ. MEASUREMENTS AND MAIN RESULTS: Stimulation of rat aortic smooth muscle cells with bacterial LPS and gamma-interferon induced the production of nitrite and nitrate (breakdown products of NO) and suppression of mitochondrial respiration for 24 to 48 hrs. The amount of NO produced was slightly enhanced with ODQ (10-100 EM), whereas the suppression of mitochondrial respiration was not affected by ODQ (1-100 microM). ODQ did not affect the degree of suppression of mitochondrial respiration in response to NO donor agents or to peroxynitrite. Exposure to LPS (10 ng/mL) for 6 hrs caused a time-dependent relaxation of norepinephrine-precontracted endothelium-denuded thoracic aortic rings. This response was caused by the expression of inducible NOS and could be blocked by pharmacologic inhibitors of NOS such as N(G)-methylL-arginine. ODQ (1 microM) prevented the LPS-induced loss of vascular tone in this experimental system. Similar to the in vitro responses, there was a significant suppression of the norepinephrine-induced contractions in ex vivo experiments, in which rings were taken from animals treated in vivo with LPS (10 mg/kg for 3 hrs). ODQ treatment in vitro (1 microM) caused a complete restoration of the contractile responses. In mice challenged with 60 mg/kg LPS ip, ODQ (20 mg/kg), given either as a pretreatment or as a 4-hr posttreatment, improved survival at 24-144 hrs. CONCLUSION: These studies indicate that GC activation does not contribute to NO- or peroxynitrite-induced cytotoxicity but does contribute to the vascular hyporeactivity induced by endotoxin in vitro and in vivo. GC inhibition alone is sufficient to influence survival in a murine model of severe sepsis.     

Insulin sensitizes neural and vascular TRPV1 receptors in the trigeminovascular system

BackgroundClinical observations suggest that hyperinsulinemia and insulin resistance can be associated with migraine headache. In the present study we examined the effect of insulin on transient receptor potential vanilloid 1 (TRPV1) receptor-dependent meningeal nociceptor functions in rats.MethodsThe effects of insulin on the TRPV1 receptor stimulation-induced release of calcitonin gene related peptide (CGRP) from trigeminal afferents and changes in meningeal blood flow were studied. Colocalization of the insulin receptor, the TRPV1 receptor and CGRP was also analyzed in trigeminal ganglion neurons.ResultsInsulin induced release of CGRP from meningeal afferents and consequent increases in dural blood flow through the activation of TRPV1 receptors of trigeminal afferents. Insulin sensitized both neural and vascular TRPV1 receptors making them more susceptible to the receptor agonist capsaicin. Immunohistochemistry revealed colocalization of the insulin receptor with the TRPV1 receptor and CGRP in a significant proportion of trigeminal ganglion neurons.ConclusionsInsulin may activate or sensitize meningeal nociceptors that may lead to enhanced headache susceptibility in persons with increased plasma insulin concentration.     

Exisulind and guanylyl cyclase C induce distinct antineoplastic signaling mechanisms in human colon cancer cells

The nonsteroidal anti-inflammatory drug sulindac is metabolized to sulindac sulfone (exisulind), an antineoplastic agent that inhibits growth and induces apoptosis in solid tumors. In colon cancer cells, the antineoplastic effects of exisulind have been attributed, in part, to induction of cyclic guanosine 3',5'-monophosphate (cGMP) signaling through inhibition of cGMP-specific phosphodiesterases, which elevates intracellular cGMP, and novel expression of cGMP-dependent protein kinase (PKG) Ibeta, the presumed downstream effector mediating apoptosis. Here, inhibition of proliferation and induction of cell death by exisulind was dissociated from cGMP signaling in human colon cancer cells. Accumulation of intracellular cGMP produced by an exogenous cell-permeant analogue of cGMP or a potent agonist of guanylyl cyclase C yielded cytostasis without cell death. Surprisingly, the antiproliferative effects of induced cGMP accumulation were paradoxically less than additive, rather than synergistic, when combined with exisulind. Further, although exisulind induced expression of PKG Ibeta, it did not elevate intracellular cGMP and its efficacy was not altered by inhibition or activation of PKG I. Rather, PKG I induced by exisulind may mediate desensitization of cytostasis induced by cGMP. Thus, cytotoxic effects of exisulind are independent of cGMP signaling in human colon cancer cells. Moreover, combination therapies, including exisulind and agents that induce cGMP signaling, may require careful evaluation in patients with colon cancer.     

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

See also Gordge MP. Nitric oxide: a one‐trick pony? This issue, pp 1340–2. Summary. 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.     

Regulation of the tracheobronchial and lung vascular system: innervation and receptors

The regulation of airway smooth muscle shows species variation. The common embryologic origin of the lung and the gut is evidenced by the fact that the morphology and function of innervation, ganglion cell population, smooth muscle cells and receptors are similar to analogous structures in the gastrointestinal tract. Human airway smooth muscle possesses the following anatomic, morphologic and functional characteristics: cholinergic excitatory nerves, non-adrenergic non-cholinergic (NANC) inhibitor nerves, non-cholinergic non-adrenergic excitatory nerves, no adrenergic nerves, an ultrastructural morphology of the ganglia, similar to the one in the gastro intestinal tract, smooth muscle cell-to-cell connection of the gap junction or nexus type, a possible spontaneous myogenic activity, Beta-2-receptors with inhibitory effect and Alpha-1-receptors with excitatory effect. The pulmonary vascular bed is a low pressure system. Pulmonary vascular tone is influenced and probably regulated by multiple neurohumoral factors. These include the autonomic nervous system and a variety of vasoactive peptides and lipides. The autonomic neurogenic influences include the alpha- and beta-adrenergic and cholinergic components, as well as a third, non-adrenergic non-cholinergic component which may be mediated by the neuropeptide Vasoactive Intestinal Polypeptide (VIP). Lung peptides - some present in neuroendocrine cells - have potent actions (relaxation or constriction) on pulmonary vessels and may participate in regulating vascular tone. Leukotriens and other biological active lipides generally constrict pulmonary vessel and promote microvascular permeability.     

α-肾上腺素受体在脊髓损伤大鼠血管高反应性中的作用

目的研究高位脊髓横切（SCT）慢性期大鼠腹主动脉离体血管环对α-肾上腺紊受体（α—AR）激动剂的反应性及腹主动脉α1-AR、α2-AR各亚型mRNA的表达量，探讨其与腹主动脉高反应性的关系。方法取SCT4周后大鼠的腹主动脉，利用离体血管环张力测定技术，以血管环对梯度浓度去氧肾上腺素（Phe）和可乐定的收缩力，反映血管环对α1-AR、α2-AR激动剂的反应性；通过实时定量聚合酶链反应（PCR）测定腹主动脉α1A—AR、α1B-AR、α1D—AR、α2A—AR、α2B—AR和α2c—AR的mRNA表达量改变，并与假手术组比较。结果与假手术组比较，SCT4周组大鼠腹主动脉对可乐定反应性上升（P〈0．05或P〈0．01），对Phe反应性无明显改变；SCT组腹主动脉α1A—AR和α1D—AR的mRNA表达上调（P〈0．05或P〈0．01），而α1B—AR mRNA表达未见明显改变（P〉0．05），α2A—AR、α2B—AR和α2c—AR的mRNA表达均上调（P〈0．05或P〈0．01）。结论在SCT慢性期，腹主动脉高反应性的机制可能为血管对α2-AR激动剂敏感性上升，α2-AR mRNA表达上调。虽然α1-AR RNA表达上调，但离体血管对Phe收缩力无明显改变，α1—AR可能并不是引起SCT慢性期血管高反应性的原因。     

Reduced serotonin vascular sensitivity in ergotamine abusers

The action of ergotamine on the 5-hydroxytryptamine (5-HT) venous sensitivity was studied in ergotamine abuser and non-abuser migraine patients. Ergotamine abusers showed reduced 5-HT hand vein contraction during abuse, compared to seven days after ergotamine withdrawal. In non-ergotamine users, the 5-HT venoconstriction was not significantly modified 12 h after a single intramuscular ergotamine (0.25 mg) administration. Even the administration of ergotamine locally into the vein did not change the venospasm of 5-HT given acutely in the same vein. Therefore, it seems that the 5-HT antagonism does not contribute to the therapeutic effect of ergotamine during the migraine attack. Moreover, the reduced 5-HT responsiveness during ergotamine abuse may possibly be compatible with the chronic headache present in some abusers, the withdrawal headache attacks and the abuse itself.     

The soluble guanylyl cyclase activator YC-1 increases intracellular cGMP and cAMP via independent mechanisms in INS-1E cells

In addition to increasing cGMP, the soluble guanylyl cyclase (sGC) activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) can elevate intracellular cAMP levels. This response was assumed to be as a result of cGMP-dependent inhibition of cAMP phosphodiesterases; however, in this study, we show that YC-1-induced cAMP production in the rat pancreatic beta cell line INS-1E occurs independent of its function as a sGC activator and independent of its ability to inhibit phosphodiesterases. This YC-1-induced cAMP increase is dependent upon soluble adenylyl cyclase and not on transmembrane adenylyl cyclase activity. We previously showed that soluble adenylyl cyclase-generated cAMP can lead to extracellular signal-regulated kinase activation and that YC-1-stimulated cAMP production also stimulates extracellular signal-regulated kinase. Although YC-1 has been used as a tool for investigating sGC and cGMP-mediated pathways, this study reveals cGMP-independent pharmacological actions of this compound.     

NO synthesis in the vascular endothelium of patients with type II diabetes

The subjects of the study were 30 patients with type II diabetes and arterial hypertension. The purpose of the study was comparative clinical characterization of type II diabetes patients with and without microalbuminuria (MAU), and evaluation of effects of 16-week therapy aimed at stimulation of NO synthesis by vascular endothelium, on the pathogenesis of diabetic nephropathy. The results show that patients with MAU, unlike those without it, are characterized by longer course of diabetes, more pronounced lipid exchange disorder, more variable arterial pressure, higher pressure load index, elevated activity of lipid peroxidation (LP) processes, prominent disorder of NO-producing endothelial function. After 16 weeks of treatment, both groups demonstrated significant increase of NO basal secretion, prominent reduction of arterial hypertension, significant improvement of important carbohydrate and lipid exchange parameters, reduction of LP activity, and thus reduction of MAU. The listed changes were more significant in patients with MAU.     

Effect of inhibition of inducible nitric oxide synthase and guanylyl cyclase on endotoxin-induced delay in gastric emptying and intestinal transit in mice

Nitric oxide (NO) is postulated to play a role in endotoxin-induced ileus. We investigated the effect of selective blockade of inducible NO synthase (iNOS) and guanylyl cyclase on endotoxin-induced ileus in mice. Thirty minutes before injection of lipopolysaccharides (LPS), mice were pretreated with L-NAME (N omega-nitro-L-arginine methyl ester, non-selective NOS inhibitor), 1400W (N-(3-(aminomethyl)benzyl)acetamide, selective iNOS inhibitor), ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, guanylyl cyclase inhibitor), dimethyl sulfoxide (DMSO, vehicle), or dexamethasone. After 18 h, general well being deteriorated and the mice developed hypothermia and a significant delay in gastric emptying and intestinal transit as measured by Evans blue. 1400W completely reversed the endotoxin-induced delay in gastric emptying, while L-NAME did not have these beneficial effects. On the contrary, even in control mice, L-NAME delayed gastric emptying. Dexamethasone, DMSO, and ODQ mimicked the effect of 1400W on endotoxin-induced delay in gastric emptying. The endotoxin-induced delay in transit was significantly improved only by 1400W. None of the drugs reversed the hypothermia. In LPS mice treated with L-NAME, the behavior scale increased even further, while it decreased after treatment with 1400W. In conclusion, selective inhibition of iNOS reverses the endotoxin-induced delay in gastric emptying and transit and improves general well being. The pathway used by NO, derived from iNOS, may involve inhibition of guanylyl cyclase or radical scavenging.     

动态脉压指数和超敏C反应蛋白与高血压患者血管硬化发生及发展

目的:分析动态脉压指数与炎症因子超敏C反应蛋白的关系,为早期防治高血压靶器官损害提供理论依据。方法:选择2004-01/2005-09在广西中医学院附属瑞康医院心内科住院的原发性高血压患者156例,均自愿参加观察。按脉压指数水平分为4组,即脉压指数≤0.40组、脉压指数0.41～0.50组、脉压指数0.51～0.60组及脉压指数>0.60组,各组分别有37,48,51及20例患者。应用全自动免疫散射比浊法检测血清超敏C反应蛋白,动态血压测定采用美国ABPM-04血压监测仪。有效血压读数标准:收缩压70～260mmHg(1mmHg=0.133kPa),舒张压40～150mmHg。监测期间要求患者坚持日常生活起居,避免剧烈活动。脉压指数为平均脉压/平均收缩压。比较各组患者的超敏C反应蛋白的水平,分析其与各血压指标之间的相关性。结果:156例患者全部进入结果分析,无脱落。①脉压指数0.41～0.50组、脉压指数0.51～0.60组及脉压指数>0.60组患者的血清超敏C反应蛋白水平均显著高于脉压指数≤0.40组[(5.62±0.76),(6.23±0.83),(6.63±0.79),(5.23±0.82)mg/L,t=2.45～6.64,P<0.05～0.01]。②超敏C反应蛋白水平与收缩压、舒张压、脉压、脉压指数呈正相关(r=0.321～0.562,P<0.05～0.01),其中以脉压指数的相关系数(r=0.562)最大。结论:原发性高血压患者的血清超敏C反应蛋白水平与动态脉压指数的关系密切,两者共同参与了动脉粥样硬化的发生发展过程。