Messenger Nitric Oxide Research Articles

Type 5 Phosphodiesterase Inhibitors in the Treatment of Erectile Dysfunction and Cardiovascular Disease

Since the discovery of sildenafil in 1989 as a highly selective inhibitor of the phosphodiesterase type-5 (PDE-5) receptor, 2 additional PDE-5 inhibitors, tadalafil and vardenafil, have emerged as safe and effective treatments of erectile dysfunction (ED). Enzymes in the PDE family catalyze the hydrolysis of the intracellular signaling molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which is the second messenger of nitric oxide (NO) and a principal mediator of smooth muscle relaxation and vasodilation. Sildenafil was initially introduced for clinical use as the result of extensive research on chemical agents targeting PDE-5 that might potentially be useful in the treatment of coronary heart disease. Erection is largely a hemodynamic event, which is regulated by vascular tone and blood flow balance in the penis. Endothelial dysfunction, an early component of atherosclerosis, may inhibit a vascular event such as erection and is rarely confined to the arteries supplying blood to the penis, but more likely occurs throughout the vascular bed. In addition to the effects of the NO-cGMP signaling pathway on cavernosal smooth muscle, clinical findings have suggested that vascular tone in the pulmonary, coronary, and other vascular tissues expressed by PDE-5 is also influenced by this signal transduction mechanism. This has led to the emergence of novel therapeutic indications for sildenafil over a range of cardiovascular conditions that are either well-established risk factors or comorbidities with ED. Recently, the U.S. Food and Drug Administration approved sildenafil as an orally active therapy for the treatment of primary pulmonary hypertension. The drug will be marketed under the trade name of Revatio, not Viagra, the name used for the ED indication. The approved dose for primary pulmonary hypertension is 20 mg 3 times daily.

Increased insulin‐stimulated endothelin‐1 release is a distinct vascular phenotype distinguishing Cushing's disease from metabolic syndrome

Although much is known about the anti-inflammatory effects of an acute corticosteroid therapy, little is known about the effects on chronic hypercortisolism on endothelial dysfunction and proinflammatory alterations in patients with Cushing's disease (CD). We studied 9 patients with CD, 10 patients with metabolic syndrome and 27 normal controls. The tests consisted of an intravenous bolus of 0.1 U/kg insulin combined with a euglycaemic clamp technique with an arterialized forearm and assessment of the training parameters deep-venous balance of forearm glucose uptake (as an index of insulin sensitivity); NO(x) (nitric oxide end-products), c-GMP (second messenger of nitric oxide) and endothelin-1 release, as indices of endothelial function and proinflammatory systemic markers. Forearm glucose uptake incremental area was significantly lower in Cushing's disease and in the metabolic syndrome than in controls, suggesting a state of severe insulin resistance. Compared to controls and to the metabolic syndrome, basal and insulin-stimulated NO(x) release incremental areas were significantly reduced in Cushing's disease, while forearm c-GMP release was similarly decreased in CD and metabolic syndrome. By contrast, endothelin-1 incremental areas after insulin bolus were significantly higher in CD than in controls and the metabolic syndrome, in the presence of increased TNF-alpha, IL-6 and CRP levels. Forearm glucose uptake incremental area significantly correlated with NO(x) incremental area, forearm c-GMP release incremental area, TNF-alpha levels and ET-1 incremental area. In patients with CD, supraphysiological insulin levels are not able to overcome the insulin resistance due to chronic hypercortisolism. Furthermore, an increased proatherogenic risk profile is characterized by decreased nitric oxide synthesis and activity, enhanced endothelin-1 levels and increased proinflammatory markers.

Nitric Oxide Regulates Input Specificity of Long-Term Depression and Context Dependence of Cerebellar Learning

Recent studies have shown that multiple internal models are acquired in the cerebellum and that these can be switched under a given context of behavior. It has been proposed that long-term depression (LTD) of parallel fiber (PF)–Purkinje cell (PC) synapses forms the cellular basis of cerebellar learning, and that the presynaptically synthesized messenger nitric oxide (NO) is a crucial “gatekeeper” for LTD. Because NO diffuses freely to neighboring synapses, this volume learning is not input-specific and brings into question the biological significance of LTD as the basic mechanism for efficient supervised learning. To better characterize the role of NO in cerebellar learning, we simulated the sequence of electrophysiological and biochemical events in PF–PC LTD by combining established simulation models of the electrophysiology, calcium dynamics, and signaling pathways of the PC. The results demonstrate that the local NO concentration is critical for induction of LTD and for its input specificity. Pre- and postsynaptic coincident firing is not sufficient for a PF–PC synapse to undergo LTD, and LTD is induced only when a sufficient amount of NO is provided by activation of the surrounding PFs. On the other hand, above-adequate levels of activity in nearby PFs cause accumulation of NO, which also allows LTD in neighboring synapses that were not directly stimulated, ruining input specificity. These findings lead us to propose the hypothesis that NO represents the relevance of a given context and enables context-dependent selection of internal models to be updated. We also predict sparse PF activity in vivo because, otherwise, input specificity would be lost.

The bladder epithelium and overactive bladder: What we know

Although the urothelium has been traditionally thought of as a passive barrier between urine and detrusor muscle, new studies have shown that the urothelium is a highly specialized structure involved in antigen presentation, micturition reflex, metabolic secretion, inflammatory regulation, and sensory afferent functioning. Data from several laboratories have shown that the urothelium can respond to thermal, mechanical, and chemical stimuli. The earlier findings (activation of urothelial transient receptor potential channel vanilloid 1 producing the second messenger nitric oxide, which in turn triggers suburothelial sensory nerves) demonstrate how the urothelium acts as a transducer, releasing chemicals that target adjacent bladder cells and sensory neurons. We now know that bladder urothelium acts also as a transducer whereby afferent neurons, via urothelial mechanoafferent transduction, are involved in the micturition process and the pathogenesis of bladder disorders. This paper highlights the important role that the urothelium has in bladder pathophysiology.

Beneficial effects of a long-term oral l-arginine treatment added to a hypocaloric diet and exercise training program in obese, insulin-resistant type 2 diabetic patients

Because chronic L-arginine supplementation improves insulin sensitivity and endothelial function in nonobese type 2 diabetic patients, the aim of this study was to evaluate the effects of a long-term oral L-arginine therapy on adipose fat mass (FM) and muscle free-fat mass (FFM) distribution, daily glucose levels, insulin sensitivity, endothelial function, oxidative stress, and adipokine release in obese type 2 diabetic patients with insulin resistance who were treated with a combined period of hypocaloric diet and exercise training. Thirty-three type 2 diabetic patients participated in a hypocaloric diet plus an exercise training program for 21 days. Furthermore, they were divided into two groups in randomized order: the first group was also treated with L-arginine (8.3 g/day), and the second group was treated with placebo. Although in the placebo group body weight, waist circumference, daily glucose profiles, fructosamine, insulin, and homeostasis model assessment index significantly decreased, L-arginine supplementation further decreased FM (P < 0.05) and waist circumference (P < 0.0001), preserving FFM (P < 0.03), and improved mean daily glucose profiles (P < 0.0001) and fructosamine (P < 0.03). Moreover, change in area under the curve of cGMP (second messenger of nitric oxide; P < 0.001), superoxide dismutase (index of antioxidant capacity; P < 0.01), and adiponectin levels (P < 0.02) increased, whereas basal endothelin-1 levels (P < 0.01) and leptin-to-adiponectin ratio (P < 0.05) decreased in the L-arginine group. Long-term oral L-arginine treatment resulted in an additive effect compared with a diet and exercise training program alone on glucose metabolism and insulin sensitivity. Furthermore, it improved endothelial function, oxidative stress, and adipokine release in obese type 2 diabetic patients with insulin resistance.

Metabolic syndrome and urinary cGMP excretion in general population

To examine the relationship between metabolic syndrome and endothelial dysfunction, we investigated cross-sectionally the correlation between metabolic risk factors and urinary excretion of cyclic guanosine 3′,5′-monophosphate (cGMP), a second messenger of nitric oxide (NO), in 1541 Japanese men and women aged 40–79 years. The 24-h urinary excretion of cGMP was measured using a 125I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The components of metabolic syndrome were defined based on the following criteria: body mass index (BMI) ≥ 25.0 kg/m 2, fasting plasma glucose ≥ 6.11 mmol/l or non-fasting plasma glucose level ≥ 11.1 mmol/l, systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg, high-density lipoprotein (HDL)-cholesterol < 1.03 mmol/l for men and <1.29 mmol/l for women, and triglyceride ≥ 1.69 mmol/l. The number of components of metabolic syndrome correlated inversely with urinary cGMP excretion; means of cGMP excretion for the whole group adjusted for age, sex, and cardiovascular risk factors were 53.6, 48.6, 47.9, 44.4 and 42.3 nmol/mmol for 0, 1, 2, 3, and 4–5 components of metabolic syndrome, respectively ( p = 0.002). Our data suggest that a reduction of NO bioactivity concur with clustered features of the metabolic syndrome.

Role of nitric oxide during neurogenesis in the olfactory epithelium

In mammals, neurogenesis continues during adulthood in restricted places of the nervous system, namely the subventricular zone, the dentate gyms and the olfactory epithelium. A dual role of the second messenger nitric oxide has been reported in such places, either promoting or inhibiting proliferation of neuronal precursors depending on the cellular signal implicated. In this review the regulation of adult olfactory epithelium neurogenesis by nitric oxide is discussed.

Enhancing cGMP in experimental progressive renal fibrosis: soluble guanylate cyclase stimulation vs. phosphodiesterase inhibition

cGMP serves as the main second messenger of nitric oxide (NO). Antifibrotic effects of enhancing renal cGMP levels have recently been documented in experimental acute anti-Thy-1 glomerulonephritis. The present study compares the effects of the cGMP production-increasing soluble guanylate cyclase (sGC) stimulator BAY 41-2272 with those of the cGMP degradation-limiting phosphodiesterase inhibitor pentoxifylline (PTX) in a progressive model of renal fibrosis. At 1 wk after induction of anti-Thy-1-induced chronic glomerulosclerosis (cGS), rats were randomly assigned to groups as follows: cGS, cGS + BAY 41-2272 (10 mg x kg body wt(-1) x day(-1)), or cGS + PTX (50 mg x kg body wt(-1) x day(-1)). BAY 41-2272 and PTX reduced systolic blood pressure significantly. At 16 wk, tubulointerstitial expressions of sGC mRNA and NO-induced cGMP synthesis were increased in untreated cGS animals, whereas their glomerular activity was depressed compared with normal controls. Tubulointerstitial and glomerular cGMP production in response to NO were significantly enhanced in animals treated with BAY 41-2272, but not in those treated with PTX. BAY 41-2272 administration resulted in marked reductions of glomerular and tubulointerstitial histological matrix accumulation, expression of TGF-beta1 and fibronectin, macrophage infiltration, and cell proliferation as well as improved renal function. In contrast, only moderate and nonsignificant renoprotective changes were observed in the cGS + PTX group. In conclusion, increasing renal cGMP production through BAY 41-2272 significantly improved renal NO-cGMP signaling and limited progression in anti-Thy-1-induced chronic renal fibrosis, whereas inhibition of cGMP degradation by PTX was only moderately effective. The findings indicate that pharmacological enhancement of renal cGMP levels by sGC stimulation represents a novel and effective antifibrotic approach in progressive kidney disorders.

Effects of selective neuronal nitric oxide synthase inhibition on sleep and wakefulness in the rat

The role played by the unconventional messenger Nitric Oxide (NO) upon the sleep–wake cycle remains controversial. Evidence suggests a positive role of NO on Slow Wave Sleep (SWS) and Paradoxical Sleep (PS) regulation, favoring sleep. However, other studies have found a role of NO upon wakefulness and alertness, inhibiting sleep. Divergences have been explained in part because of the use of different inhibitors of nitric oxide synthases (NOS). The aim of this study is to analyse the effects of a highly selective neuronal NOS inhibitor (3-Bromo7-Nitroindazole) on sleep–wake states in rats. Male Wistar rats were stereotaxically prepared for polysomnography. 3-Bromo-7-Nitroindazole (10, 20, 40 mg/kg, i.p.) dissolved in DMSO 10% filled with saline, or vehicle (DMSO 10% in saline) was administered at the beginning of the light period. Three hours of polygraphic recordings were evaluated for stages of vigilance. Results show dose-dependent effects of 3-Bromo7-Nitroindazole upon sleep: 10 mg/kg decreases duration and number of episodes of deep SWS, increasing duration of light SWS. 20 mg/kg decreased duration of light and deep SWS, while active and quiet wake increased. Deep SWS and PS latency increased. Number of episodes of PS decreased, as well as number of cycles of sleep and time spent asleep. 40 mg/kg reduced duration of deep SWS and increased mean episode duration of light SWS. Therefore, sleep states are affected by selective inhibition of nNOS, reducing in all cases deep SWS. These results support the hypothesis that nitric oxide, produced by nNOS, is involved in sleep processes, favoring sleep.

Nitric oxide, ceramide and sphingomyelinase-coupled receptors: A tale of enzymes and messengers coordinating cell death, survival and differentiation

The gaseous messenger nitric oxide plays a role in a variety of biological functions. Evidence accumulated over the last 7 years indicates that functions of nitric oxide in apoptosis growth and differentiation may originate in part from an interplay with signalling members of the sphingolipid family. Interactions between nitric oxide and sphingolipids have been shown to be multiple, to involve regulation of activity and expression of the enzymes responsible for the synthesis of nitric oxide and of those involved in the sphingolipid metabolic pathways. Recent evidence indicates that one of these interactions, namely the cross-talk of sphingomyelinases and their product ceramide with nitric oxide and its generating enzyme endothelial nitric oxide synthase, plays prominent roles during key patho-physiological processes such as inflammation, proliferation, death and differentiation.

A Cellular System to Study the Role of Nitric Oxide in Cell Death, Survival, and Migration

The gaseous messenger nitric oxide (NO) plays a bewildering number of roles in fundamental processes, such as cell locomotion, differentiation, proliferation and death. Its different and often contrasting roles may depend on its concentration and intracellular site of generation. We describe here a simple system with which to investigate the roles of NO generated at physiological levels in HeLa cells by eNOS transfected under an inducible promoter. This system has allowed us to uncover unexpected signalling circuits between NO and ceramide, involved in the response of cells to apoptotic stimuli. At present, we are using these cells as a tool to investigate the role of NO in migration.

Dimerization of Nitric Oxide-sensitive Guanylyl Cyclase Requires the α1 N Terminus

Dimerization of Nitric Oxide-sensitive Guanylyl Cyclase Requires the α1 N Terminus

STOP and GO with NO: Nitric oxide as a regulator of cell motility in simple brains

During the formation of the brain, neuronal cell migration and neurite extension are controlled by extracellular guidance cues. Here, I discuss experiments showing that the messenger nitric oxide (NO) is an additional regulator of cell motility. NO is a membrane permeant molecule, which activates soluble guanylyl cyclase (sGC) and leads to the formation of cyclic GMP (cGMP) in target cells. The analysis of specific cells types in invertebrate models such as molluscs, insects and the medicinal leech provides insight how NO and cyclic nucleotides affect the wiring of nervous systems by regulating cell and growth-cone motility. Inhibition of the NOS and sGC enzymes combined with rescue experiments show that NO signalling orchestrates neurite outgrowth and filopodial dynamics, cell migration of enteric neurons, glial migration and axonogenesis of pioneer fibers. Cultured insect embryos are accessible model systems in which cellular mechanisms of NO-induced cytoskeletal reorganizations can be analyzed in natural settings. Finally, I will outline some indications that NO may also regulate cell motility in the developing and regenerating vertebrate nervous system.

Residues stabilizing the heme moiety of the nitric oxide sensor soluble guanylate cyclase

Soluble guanylate cyclase, a heterodimer consisting of an α- and a heme-containing β-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine 105 and the recently identified counterparts of the heme propionic acids, tyrosine 135 and arginine 139. The latter residues together with an invariant serine 137 form the unique heme binding motif Y-x- S-x- R. In this work, we show that replacement of the serine 137 with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.

Expression of the protein inhibitor of nitric oxide synthase in the adult rat retina before and after optic nerve lesion

The molecular messenger nitric oxide (NO) not only serves a number of physiologic functions, but is also involved in the pathophysiology of neurodegeneration. It is produced by the nitric oxide synthase (NOS) isoenzymes. One of the many players regulating NOS activity is the Protein Inhibitor of NOS, PIN. To gain further insight into the mechanisms of NOS regulation and NO-mediated cell death after nerve trauma, we examined PIN expression in a standard model of lesion-induced neurodegeneration, the rat optic nerve transsection model. In both the axotomized retinae and the control retinae PIN expression was predominantly observed in the retinal ganglion cell layer. Optic nerve lesion did neither change the amount of PIN mRNA, as determined by in situ hybridization and real-time RT-PCR, nor did it change the amount of PIN as determined by immunohistochemistry and Western blot analysis. These results suggest that in our model, NOS activity is not regulated by altered PIN levels, which contributes to our understanding of apoptotic mechanisms in injured neurons.

Nitric Oxide-Directed Synaptic Remodeling in the Adult Mammal CNS

In adult mammals, learning, memory, and restoration of sensorimotor lost functions imply synaptic reorganization that requires diffusible messengers-mediated communication between presynaptic and postsynaptic structures. A candidate molecule to accomplish this function is the gaseous intercellular messenger nitric oxide (NO), which is involved in synaptogenesis and projection refinement during development; however, the role of NO in synaptic reorganization processes in adulthood remains to be established. In this work, we tested the hypothesis that this free radical is a mediator in the adult mammal CNS synaptic remodeling processes using a model of hypoglossal axonal injury recently developed by us. Axonal injury-induced disconnection of motoneurons from myocytes produces withdrawal of synaptic inputs to motoneurons and concomitant upregulation of the neuronal isoform of NO synthase (NOS-I). After recovery of the neuromuscular function, synaptic coverage is reestablished and NOS-I is downregulated. We also report, by using functional and morphological approaches, that chronic inhibition of the NO/cGMP pathway prevents synaptic withdrawal evoked by axon injury, despite the persistent muscle disconnection. After successful withdrawal of synaptic boutons, inhibition of NO synthesis, but not of cGMP, accelerated the recovery of synaptic coverage, although neuromuscular disconnection was maintained. Furthermore, protein S-nitrosylation was upregulated after nerve injury, and this effect was reversed by NOS-I inhibition. Our results suggest that during synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMP-dependent and probably S-nitrosylation-mediated mechanisms, respectively. We also suggest a feasible role of NO in neurological disorders coursing with NOS-I upregulation.

Relationship between urinary cGMP excretion and serum total cholesterol levels in a general population

Hypercholesterolemia impairs endothelial function. However, the critical level of serum total cholesterol at which endothelial dysfunction occurs is unknown at present. We investigated cross-sectionally the correlation between urinary excretion of cyclic guanosine 3′,5′ monophosphate (cGMP), a second messenger of nitric oxide (NO) and serum total cholesterol concentrations in a general population sample of Japanese men and women. The samples comprised 1541 subjects (788 men and 753 women) aged 40–79 years, who participated in cardiovascular risk surveys between 1997 and 2002 and underwent a 24 h urine collection. Urinary excretion of cGMP was measured using a 125I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The mean urinary cGMP excretion correlated linearly and inversely with serum total cholesterol level: mean cGMP excretion adjusted for age, sex and cardiovascular risk factors was 61.7, 53.6, 50.8, 49.2, 47.3 and 46.4 nmol/mmol for total cholesterol levels <4.14, 4.14–4.64, 4.65–5.16, 5.17–5.68, 5.69–6.20 and ≥6.21 mmol/L, respectively ( p = 0.007). This relation was more evident among individuals with end-organ damage, among subjects with higher C-reactive protein (CRP) levels and among postmenopausal women. Our data suggest a reduction of NO bioactivity with higher serum total cholesterol levels, even within clinically normal cholesterol levels.

Importance of NO/cGMP signalling via cGMP‐dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol

Cyclic GMP is a second messenger for nitric oxide (NO) that acts as a mediator for many different physiological functions. The cGMP-dependent protein kinases (cGKs) mediate cellular signalling induced by NO and cGMP. Here, we explored the localization of cGMP-dependent protein kinase type II (cGKII) in the mouse brain. In situ hybridization revealed high levels of cGKII mRNA in cerebral cortex, thalamic nuclei, hypothalamic nuclei, and in several basal forebrain regions including medial septum, striatum and amygdala. The close link to NO and the distribution pattern of cGKII suggested that this enzyme might be involved in emotional reactions and responses to drugs of abuse. Therefore, cGKII knockout animals (cGKII-/-) were compared with littermate controls in behavioural tests (i) for emotion-linked and (ii) for acute and chronic ethanol responses. Deletion of cGKII did not influence aggressive behaviour but led to enhanced anxiety-like behaviour. In terms of acute responses to ethanol, cGKII-/- mice were hyposensitive to hypnotic doses of ethanol as measured by the loss of righting reflex, without an alteration in their blood alcohol elimination. In a two-bottle free choice test, cGKII-/- mice showed elevated alcohol consumption. No taste differences to sweet solutions were observed compared to control animals. In summary, our data show that cGKII activity modulates anxiety-like behaviour and neurobehavioural effects of alcohol.

The Case for a Subcutaneous Magnesium Product and Delivery Device for Space Missions

Cardiovascular (CV) complications, associated with space flight (SF), are caused by microgravity, hypokinesia and radiation, particularly beyond earth orbit, with all three conducive to oxidative stress. Except for emergencies, pharmaceuticals appear to be contraindicated, because of unpredictable side effects from malabsorption (M) and potential hepatic and renal impairment. Magnesium (Mg) depletion and elevations of cytokines (interleukin 6) occur during SF, conducive to self-sustaining vascular inflammation mechanisms. There are potential endothelial injuries (EI) and reduced Cyclic GMP (a second messenger of nitric oxide: NO) and elevated urinary excretion of C-peptide (insulin resistance: IR). Recent findings that show reductions in vascular endothelial growth factor (VEGF) suggest that this may result from SF-related thrombocytopenia since platelets (P) are the major source of VEGF, and that NO might play a role. Both VEGF and Mg are vital for angiogenesis, endothelial function and reendothelialization. Insulin is necessary for VEGF expression. To prevent SF-related CV complications in the presence of IR and M and with the potential for renal insufficiency, closely monitored subcutaneous (SC) Mg should be provided. The dosage can be monitored by sublingual intracellular Mg assays. Needed is development of a SC Mg reservoir device, which can be replenished before extra-vehicular activities (EVA) and which must be reliable despite vigorous movements during EVA, that can last up to 8 hours. This could also be protective against decompression sickness and EVA-related 100% oxygen requirements before and during this activity, both of which predispost to EI.

Distribution of soluble guanylyl cyclase in the rat brain.

The diffusible messenger nitric oxide (NO) acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprising alpha and beta subunits. We used immunohistochemistry to study the distribution of both sGC subunits in the brain of adult rats. alpha and beta subunits gave similar widespread staining throughout the CNS, which was strongest in neostriatum, olfactory tubercle, and supraoptic nucleus. Double-labeling experiments showed striking cellular colocalization in most brain regions, suggesting that the two subunits may be organized into enzymatically active alpha/beta heteromers. Mismatches were observed in cerebellar cortex: Purkinje cells and Bergmann glia were positive for both subunits, whereas granule cells and interneurons in the molecular layer were strongly immunopositive for beta but only weakly stained for the alpha subunit. By using multiple labeling, we compared the localization of sGC with neuronal nitric oxide synthase (NOS-I, the NO-producing enzyme in neurons). In forebrain, the distribution of sGC and NOS-I was complementary, with only occasional colocalization. In contrast, cellular colocalization was common in midbrain and cerebellum. These data support a widespread role for the NO/sGC/cGMP pathway in the CNS and suggest that, in addition to its role as paracrine messenger, NO may also be an intracellular autocrine agent.