Clinical relevance of cyclic GMP modulators: A translational success story of network pharmacology.

Therapies that modulate cyclic guanosine-3'-5'-monophosphate (cGMP) have emerged as one of the most successful areas in recent drug discovery and clinical pharmacology. Historically, their focus has been on cardiovascular disease phenotypes; however, cGMP's relevance is likely to go beyond this rather limited organ-based set of indications. Moreover, the multitude of targets and their apparent interchangeability is a proof-of-concept of network pharmacology.

C-type natriuretic peptide signalling drives homeostatic effects in human chondrocytes.

Signals induced by mechanical loading and C-type natriuretic peptide (CNP) represent chondroprotective routes that may potentially prevent osteoarthritis (OA). We examined whether CNP will reduce hyaluronan production and export via members of the multidrug resistance protein (MRP) and diminish pro-inflammatory effects in human chondrocytes. The presence of interleukin-1ß (IL-1ß) increased HA production and export via MRP5 that was reduced with CNP and/or loading. Treatment with IL-1ß conditioned medium increased production of catabolic mediators and the response was reduced with the hyaluronan inhibitor, Pep-1. The induction of pro-inflammatory cytokines by the conditioned medium was reduced by CNP and/or Pep-1, αCD44 or αTLR4 in a cytokine-dependent manner, suggesting that the CNP pathway is protective and should be exploited further.

Role of C-type natriuretic peptide signalling in maintaining cartilage and bone function.

C-type natriuretic peptide (CNP) has been demonstrated in human and mouse models to play critical roles in cartilage homeostasis and endochondral bone formation. Indeed, targeted inactivation of the genes encoding CNP results in severe dwarfism and skeletal defects with a reduction in growth plate chondrocytes. Conversely, cartilage-specific overexpression of CNP was observed to rescue the phenotype of CNP deficient mice and significantly enhanced bone growth caused by growth plate expansion. In vitro studies reported that exogenous CNP influenced chondrocyte differentiation, proliferation and matrix synthesis with the response dependent on CNP concentration. The chondroprotective effects were shown to be mediated by natriuretic peptide receptor (Npr)2 and enhanced synthesis of cyclic guanosine-3',5'-monophosphate (cGMP) production. Recent studies also showed certain homeostatic effects of CNP are mediated by the clearance inactivation receptor, Npr3, highlighting several mechanisms in maintaining tissue homeostasis. However, the CNP signalling systems are complex and influenced by multiple factors that will lead to altered signalling and tissue dysfunction. This review will discuss the differential role of CNP signalling in regulating cartilage and bone homeostasis and how the pathways are influenced by age, inflammation or sex. Evidence indicates that enhanced CNP signalling may prevent growth retardation and protect cartilage in patients with inflammatory joint disease.

Intrinsic defence capacity and therapeutic potential of natriuretic peptides in pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder. EXPERIMENTAL APPROACH: Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro. KEY RESULTS: Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFß-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients. CONCLUSIONS AND IMPLICATIONS: These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.

Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia-reperfusion injury in mice.

Nitrite is protective against renal ischemia/reperfusion injury (IRI); an effect due to its reduction to nitric oxide (NO). In addition to other reductase pathways, endothelial NO synthase (eNOS) may also facilitate nitrite reduction in ischemic environments. We investigated the role of eNOS in sodium nitrite (60 microM, 10 ml/kg applied topically 1 min before reperfusion)-induced protection against renal IRI in C57/BL6 wild-type (WT) and eNOS knockout (eNOS KO) mice subjected to bilateral renal ischemia (30 min) and reperfusion (24h). Markers of renal dysfunction (plasma [creatinine] and [urea]), damage (tubular histology) and inflammation (cell recruitment) were elevated following IRI in WT mice; effects significantly reduced following nitrite treatment. Chemiluminescence analysis of cortical and medullary sections of the kidney demonstrated rapid (within 1 min) distribution of nitrite following application. Whilst IRI caused a significant (albeit substantially reduced compared to WT mice) elevation of markers of renal dysfunction and damage in eNOS KO mice, the beneficial effects of nitrite were absent or reduced, respectively. Moreover, nitrite treatment enhanced renal dysfunction in the form of increased plasma [creatinine] in eNOS KO mice. Confirmation of nitrite reductase activity of eNOS was provided by demonstration of nitrite (100 microM)-derived NO production by kidney homogenates of WT mice, that was significantly reduced by L-NMMA. L-NMMA was without effect using kidney homogenates of eNOS KO mice. These results support a role for eNOS in the pathways activated during renal IRI and also identify eNOS as a nitrite reductase in ischemic conditions; activity which in part underlies the protective effects of nitrite.

Reciprocal regulation of human soluble and particulate guanylate cyclases in vivo.

BACKGROUND & PURPOSE: We demonstrated previously that reciprocal regulation of soluble (sGC) and particulate (pGC) guanylate cyclases by NO and natriuretic peptides coordinates cyclic cGMP-mediated vasodilatation in vitro. Herein, we investigated whether such an interaction contributes to vascular homeostasis in mice and humans in vivo. EXPERIMENTAL APPROACH: Mean arterial blood pressure (MABP) changes in anaesthetized mice were monitored in response to i.v. administration of cGMP- and cAMP-dependent vasodilators in wild-type (WT), endothelial NO synthase (eNOS) and natriuretic peptide receptor (NPR)-A knockout mice. Forearm blood flow (FBF) in response to intra-brachial infusion of ANP (25, 50, 100, 200 pmol min(-1)) in the absence and presence of the NOS inhibitor NG-methyl-L-arginine (L-NMA; 4 micromol min(-1)) and the control constrictor noradrenaline (240 pmol min(-1)) was assessed in healthy volunteers. KEY RESULTS: Sodium nitroprusside (SNP; NO-donor) and atrial natriuretic peptide (ANP) produced dose-dependent reductions in MABP in WT animals that were significantly enhanced in eNOS KO mice. In NPR-A K mice, SNP produced a dose-dependent reduction in MABP that was significantly greater than that in WT mice. Responsiveness to the cAMP-dependent vasodilator epoprostenol was similar in WT, eNOS KO and NPR-A KO animals. ANP caused vasodilatation of the forearm resistance vasculature that was significantly greater in individuals lacking endothelium-derived NO (i.e. L-NMA treated). CONCLUSIONS & IMPLICATIONS: These data demonstrate that crosstalk occurs between the NO-sGC and ANP-pGC pathways to regulate cGMP-dependent vasodilatation in vivo in both mice and humans. These findings have implications for understanding the link between natriuretic peptide activity and cardiovascular risk.

Novel aspects of endothelium-dependent regulation of vascular tone.

The vascular endothelium plays a crucial role in the regulation of vascular homeostasis and in preventing the initiation and progress of cardiovascular disease by controlling mechanical functions of the underlying vascular smooth muscle. Three vasodilators: nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor, produced by the endothelium, underlie this activity. These substances act in a co-ordinated interactive manner to maintain normal endothelial function and operate as support mechanisms when one pathway malfunctions. In this review, we discuss recent advances in our understanding of how gender influences the interaction of these factors resulting in the vascular protective effects seen in pre-menopausal women. We also discuss how endothelial NO synthase (NOS) can act in both a pro- and anti-inflammatory action and therefore is likely to be pivotal in the initiation and time course of an inflammatory response, particularly with respect to inflammatory cardiovascular disorders. Finally, we review recent evidence demonstrating that it is not solely NOS-derived NO that mediates many of the beneficial effects of the endothelium, in particular, nitrite acts as a store of NO released during pathological episodes associated with NOS inactivity (ischemia/hypoxia). Each of these more recent findings has emphasized new pathways involved in endothelial biology, and following further research and understanding of the significance and mechanisms of these systems, it is likely that new and improved treatments for cardiovascular disease will result.

C-type natriuretic peptide: new candidate for endothelium-derived hyperpolarising factor.

Endothelium-derived hyperpolarising factor (EDHF) is an important regulator of vascular tone; however, its identity is still unclear. Several different molecules have been suggested, the most recent of which is the 22-amino acid peptide C-type natriuretic peptide (CNP). CNP induces hyperpolarisation and relaxation of rat mesenteric resistance artery vascular smooth muscle through activation of natriuretic peptide receptor subtype C (NPR-C) and the same potassium channels as EDHF. In addition, this peptide is released from endothelial cells of the perfused rat mesenteric bed in response to endothelium-dependent vasodilators. Thus, CNP is likely to play a vital role in regulation of vascular tone. In addition, since there is evidence that up-regulation of EDHF occurs where normal endothelium function has been compromised, modulation of this pathway represents a novel target for therapeutics in the treatment of inflammatory cardiovascular pathologies characterised by endothelial dysfunction.

Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide.

Expression of inducible NO synthase (iNOS) by macrophages is a prerequisite for the production of high output NO, which mediates many bactericidal and tumoricidal actions of these immune cells. The expression of iNOS in mammalian cells is governed predominantly by the transcription factor, NF-kappa B, which regulates the expression of many host defense proteins. In the present study, we characterize a novel, biphasic effect of NO on NF-kappa B activity in murine macrophages. This mechanism depends on the local concentration of NO and enables it both to up- and down-regulate the expression of host defense proteins including iNOS, cyclooxygenase-2, and IL-6. This biphasic activity of NO appears to play a pivotal role in the time course of activation of these immune cells and, by inference, in facilitating the initiation of a defense response against pathogenic stimuli and in its termination to limit tissue damage. This mechanism may explain at least in part the reported ability of NO to act in both a pro- and anti-inflammatory manner.

Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases.

Nitric oxide (NO) and atrial natriuretic peptides (ANP) activate soluble (sGC) and particulate guanylate cyclase (pGC), respectively, and play important roles in the maintenance of cardiovascular homeostasis. However, little is known about potential interactions between these two cGMP-generating pathways. Here we demonstrate that sGC and pGC cooperatively regulate cGMP-mediated relaxation in human and murine vascular tissue. In human vessels, the potency of spermine-NONOate (SPER-NO) and ANP was increased after inhibition of endogenous NO synthesis and decreased by prior exposure to glyceryl trinitrate (GTN). Aortas from endothelial NO synthase (eNOS) knockout (KO) mice were more sensitive to ANP than tissues from wild-type (WT) animals. However, in aortas from WT mice, the potency of ANP was increased after pretreatment with NOS or sGC inhibitor. Vessels from eNOS KO animals were less sensitive to ANP after GTN pretreatment, an effect that was reversed in the presence of an sGC inhibitor. cGMP production in response to SPER-NO and ANP was significantly greater in vessels from eNOS KO animals compared with WT animals. This cooperative interaction between NO and ANP may have important implications for human pathophysiologies involving deficiency in either mediator and the clinical use of nitrovasodilators.

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

Autoregulation of nitric oxide-soluble guanylate cyclase-cyclic GMP signalling in mouse thoracic aorta.

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.

Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity.

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanisms responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives have been synthesized and characterized. HbO(2) can be nitrosated at up to three distinct residues, one in the alpha-globin chain and two in the beta-chain. A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-dependent mechanism involving nucleophilic attack at the nitrosated thiol functionality of SNO-Hb; in the case of glutathione, this process was associated with the formation of a mixed disulfide. In contrast, multinitrosated hemoglobin species released NO and relaxed vascular smooth muscle by a thiol-independent mechanism. HbO(2) scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative intermediate; the presence of a low molecular weight thiol is a prerequisite for this process. Such observations have important implications for the generation, metabolic fate, and biological activity of S-nitrosothiols.

Comparison of sevoflurane and total intravenous anaesthesia for daycase urological surgery.

Target-controlled total intravenous anaesthesia using propofol and remifentanil was compared with inhalational anaesthesia using sevoflurane and alfentanil in patients undergoing daycase urological surgery. Seventy-one patients were randomly allocated to receive either a target-controlled infusion of an admixture of propofol and remifentanil (125 microg of remifentanil added to 500 mg of propofol), or inhalational anaesthesia with sevoflurane and intra-operative alfentanil. There was no difference in time to fitness for discharge, time to fitness for transfer from primary to secondary recovery, time to first oral intake or adverse anaesthetic induction occurrences. Patient satisfaction assessed at 24 h post-discharge was high in both groups with no significant difference between groups. The incidence of nausea and vomiting was low in both groups. We conclude that, for ultra-short stay surgery, both the techniques we describe offer satisfactory anaesthesia with very early resumption of street fitness.

Inhibition of nitric oxide synthase as a potential therapeutic target.

Nitric oxide (NO) regulates numerous physiological processes, including neurotransmission, smooth muscle contractility, platelet reactivity, and the cytotoxic activity of immune cells. Because of the ubiquitous nature of NO, inappropriate release of this mediator has been linked to the pathogenesis of a number of disease states. This provides the rationale for the design of therapies that modulate NO concentrations selectively. A well-characterized family of compounds are the inhibitors of NO synthase, the enzyme responsible for the generation of NO; such agents are potentially beneficial in the treatment of conditions associated with an overproduction of NO, including septic shock, neurodegenerative disorders, and inflammation. This article provides an overview of NO synthase inhibitors, focusing on agents that prevent binding of substrate L-arginine.

Soluble guanylate cyclase: the forgotten sibling.

Despite widespread distribution in most mammalian cells, the role of soluble guanylate cyclase has, until recently, been poorly defined, especially when compared with its more illustrious sibling, adenylate cyclase. In this review Adrian Hobbs outlines some of the reasons why the soluble guanylate cyclase-cGMP pathway has remained outside the signalling spotlight for much of the past 30 years. He goes on to describe how new molecular biological and biochemical approaches have facilitated a characterization of soluble guanylate cyclase and how this enzyme has acquired a profound physiological significance, and much research attention, as the intracellular 'receptor' for nitric oxide.

Nitric oxide regulates endothelium-dependent vasodilator responses in rabbit hindquarters vascular bed in vivo.

The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelium-dependent vasodilation in vivo. To this end, the influence of exogenous NO on vasodilator responses in the rabbit hindquarters vascular bed was determined. Previous in vitro studies have demonstrated that NO inhibits both neuronal NO synthase from rat cerebellum as well as NO synthase derived from bovine aortic endothelial cells. The present study was conducted in the rabbit hindquarters vascular bed under conditions of constant blood flow so that changes in pressure directly reflected changes in vascular resistance. Under these in vivo conditions, the NO donor agent S-nitroso-N-acetylpenicillamine (SNAP) reversibly attenuated responses to the endothelium-dependent vasodilators, acetylcholine and bradykinin. In contrast, SNAP did not influence the endothelium-independent vasodilator response to SNAP itself or to 8-bromoguanosine 3',5'-cyclic monophosphate. These observations indicate clearly that NO interferes with endothelium-dependent vasodilator action and support the view that endogenous NO may actually play a physiological role in regulating vascular tone.