Nitric Oxide-cyclic Guanosine Monophosphate Research Articles

Methylene Blue for Distributive Shock: A Potential New Use of an Old Antidote

Methylene blue is used primarily in the treatment of patients with methemoglobinemia. Most recently, methylene blue has been used as a treatment for refractory distributive shock from a variety of causes such as sepsis and anaphylaxis. Many studies suggest that the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway plays a significant role in the pathophysiology of distributive shock. There are some experimental and clinical experiences with the use of methylene blue as a selective inhibitor of the NO-cGMP pathway. Methylene blue may play a role in the treatment of distributive shock when standard treatment fails.

Volume overload induces differential spatiotemporal regulation of myocardial soluble guanylyl cyclase in eccentric hypertrophy and heart failure

Nitric oxide activation of soluble guanylyl cyclase (sGC) blunts the cardiac stress response, including cardiomyocyte hypertrophy. In the concentric hypertrophied heart, oxidation and re-localization of myocardial sGC diminish cyclase activity, thus aggravating depressed nitric oxide–cyclic guanosine monophosphate (NO–cGMP) signaling in the pressure-overloaded failing heart. Here, we hypothesized that volume-overload differentially disrupts myocardial sGC activity during early compensated and late decompensated stages of eccentric hypertrophy. To this end, we studied the expression, redox state, subcellular localization, and activity of sGC in the left ventricle of dogs subjected to chordal rupture-induced mitral regurgitation (MR). Unoperated dogs were used as Controls. Animals were studied at 4weeks and 12months post chordal rupture, corresponding with early (4wkMR) and late stages (12moMR) of eccentric hypertrophy. We found that the sGC heterodimer subunits relocalized away from caveolae-enriched lipid raft microdomains at different stages; sGCβ1 at 4wkMR, followed by sGCα1 at 12moMR. Moreover, expression of both sGC subunits fell at 12moMR. Using the heme-dependent NO donor DEA/NO and NO-/heme-independent sGC activator BAY 60-2770, we determined the redox state and inducible activity of sGC in the myocardium, within caveolae and non-lipid raft microdomains. sGC was oxidized in non-lipid raft microdomains at 4wkMR and 12moMR. While overall DEA/NO-responsiveness remained intact in MR hearts, DEA/NO responsiveness of sGC in non-lipid raft microdomains was depressed at 12moMR. Caveolae-localization protected sGC against oxidation. Further studies revealed that these modifications of sGC were also reflected in caveolae-localized cGMP-dependent protein kinase (PKG) and MAPK signaling. In MR hearts, PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) disappeared from caveolae whereas caveolae-localization of phosphorylated ERK5 increased. These findings show that differential oxidation, re-localization, and expression of sGC subunits distinguish eccentric from concentric hypertrophy as well as compensated from decompensated heart failure.

The Effects of Watermelon (Citrullus lanatus) Extracts and l-Citrulline on Rat Uterine Contractility

In uterine smooth muscle, the effects of watermelon and its citrulline content are unknown. The aims of this study were therefore, to determine the effects of watermelon extract and citrulline on the myometrium and to investigate their mechanisms of action. The effects of extracts of watermelon flesh and rind and L-citrulline (64 μmol/L) were evaluated on 3 types of contractile activity; spontaneous, those elicited by potassium chloride (KCl) depolarization, or oxytocin (10 nmol/L) application in isolated rat uterus. Inhibitors of nitric oxide (NO) and its mechanisms of action, N ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μmol/L), LY83583 (1 μmol/L), and tetraethylamonium chloride (5 mmol/L), as well as Ca signaling pathways, were determined. Both flesh and rind extracts significantly decreased the force produced by all 3 mechanisms, in a dose-dependent manner. The extracts could also significantly decrease the force under conditions of sustained high Ca levels (depolarization and agonist) and when the force was produced only by sarcoplasmic reticulum (SR) Ca release. L-citrulline produced the same effects on force as watermelon extracts. With submaximal doses of extract, the additive effects of L-citrulline were found. The inhibitory effects of extracts and L-citrulline were reversed upon the addition of NO inhibitors, and pretreatment of tissues with these inhibitors prevented the actions of both extracts and L-citrulline. Thus, these data show that watermelon and citrulline are potent tocolytics, decreasing the force produced by calcium entry and SR release and arising by different pathways, including oxytocin stimulation. Their major mechanism is to stimulate the NO-cyclic guanosine monophosphate (cGMP) relaxant pathway.

SaRNA Guided iNOS Up-Regulation Improves Erectile Function of Diabetic Rats

Promoter targeted saRNAs mediate sequence specific up-regulation of gene expression. We explored the therapeutic effect of RNA activation mediated iNOS gene activation on improving erectile function in a rat model of diabetes mellitus. An optimal saRNA sequence specific for iNOS promoter was cloned into an adenoviral vector, resulting in AdU6/shiNOS and AdU6/shControl. The corresponding viruses were used to transduce cultured rat cavernous smooth muscle cells. Streptozotocin induced diabetes models were established in rats and used to test the effects of intracavernous delivery of iNOS saRNA viruses on erectile function. iNOS expression in the cavernous smooth muscle cells or penile tissue of treated rats was assessed by reverse transcriptase-polymerase chain reaction and Western blot. Cyclic guanosine monophosphate was analyzed by enzyme-linked immunosorbent assay. Intracavernous pressure in response to cavernous nerve stimulation was measured using a data acquisition system on post-injection days 1, 3, 5, 7, 10 and 14. Adenovirus mediated expression of iNOS saRNA caused sustained up-regulation of iNOS in cavernous smooth muscle cells. Intracavernous injection of AdU6/shiNOS activated iNOS expression in vivo and significantly increased peak intracavernous pressure in streptozotocin induced diabetic rats via nitric oxide/intracellular cyclic guanosine monophosphate activation. Results show that saRNA mediated iNOS over expression in the penis can restore erectile function in streptozocin diabetic rats via the nitric oxide-cyclic guanosine monophosphate pathway.

Modulation of paracetamol-induced hepatotoxicity by phosphodiesterase isozyme inhibition in rats: a preliminary study

Altered regulation of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) is present in liver cirrhosis. Several experimental studies have shown that selective modulation of NO metabolism in the liver reduces intrahepatic resistance and portal pressure in cirrhosis. This preliminary study investigated whether selective inhibition of phosphodiesterase-5 (PDE-5), which prevents the conversion of cGMP to 5'-GMP, as well as non-selective inhibition of PDE isozymes could ameliorate hepatic toxicity induced by paracetamol (PCM). PCM (250 mg/kg, i.p.) was administered to induce hepatotoxicity. Control rats received physiological saline (10 mL/kg, p.o.), while sildenafil (a selective PDE-5 inhibitor) and aminophylline (a non-selective PDE inhibitor) were administered separately at 10 mg/kg p.o. to PCM-treated rats. PCM hepatotoxicity, characterized by elevation of aspartate and alanine aminotransferases, hepatic degeneration, and centrilobular necrosis, was attenuated by both PDE inhibitors. Sildenafil and aminophylline significantly (p<0.05) reduced plasma aspartate aminotransferase activity by 49.6% and 39.8%, respectively, with moderate increase in alanine aminotransferase activity by 26.1% and 20.4%, respectively, in PCM-treated rats. Decreases in total protein and albumin induced by PCM were significantly (p<0.05) prevented by 30.0% and 22.2%, respectively, following sildenafil administration, while aminophylline decreased these proteins by 14.0% and 25.9%, respectively. Sildenafil and aminophylline significantly (p<0.05) reduced lipid peroxidation by 30.7% and 19.7%, respectively, while moderately increasing glutathione (GSH) in the PCM-treated rats. Both drugs did not significantly alter the total cholesterol and triglyceride levels. These preliminary data suggest that pharmacological inhibition of PDE isozymes may be a useful strategy in protecting against PCM hepatic toxicity.

Latest treatment for lower urinary tract dysfunction: Therapeutic agents and mechanism of action

Recent studies suggest that antimuscarinics might suppress bladder afferent activity by blocking muscarinic receptors in the urothelium, myofibroblasts and detrusor, thereby improving overactive bladder symptoms. β(3)-Adrenoceptors are predominantly expressed in the human bladder and mediate relaxation of detrusor muscle. β(3)-Adrenoceptor agonists increase bladder capacity and prolong micturition interval. It is assumed that β(3)-adrenoceptor agonists could exert an inhibitory effect on bladder afferent through β(3)-adrenoceptors in the urothelium and detrusor, which eventually improve the symptom of urgency. Mirabegron is a potent and selective β(3)-adrenoceptor agonist. A Japanese phase 3 study showed that mirabegron has excellent efficacy and safety for treating overactive bladder. α(1)-Adrenoceptor antagonists (α(1)-blockers) have become a mainstay of male lower urinary tract symptoms treatment. The α(1)(A) subtype is known to mediate functional obstruction as a result of benign prostatic enlargement. Recent studies have suggested that α(1)(A)-adrenoceptors are additionally involved in the generation of storage symptoms. The α(1)(D) subtype is thought to play a role in the facilitation of voiding reflex; that is; storage symptoms. α(1)-Blockers often fail to alleviate overactive bladder symptoms. In this context, combination therapy with α(1)-blockers and antimuscarinics has been recommended. Treatment with 5α-reductase inhibitor for 1 year improves urinary symptoms and flow rate by reducing prostatic volume in men with benign prostatic enlargement. A pooled analysis showed that the long-term (2 or 4 years) treatment with 5α-reductase inhibitor reduced the rate of progression to acute urinary retention and surgery. Combination therapy with 5α-reductase inhibitor and α(1)-blocker was shown to provide a rapid improvement in lower urinary tract symptoms, and reduce the relative risk of acute urinary retention and benign prostatic hyperplasia-related surgery. Phosphodiesterase inhibitors might target a nitric oxide-cyclic guanosine monophosphate pathway in the prostate, urethra and bladder. Phosphodiesterase-5 inhibitors (sildenafil or tadalafil) were shown to provide clinically relevant improvements in both male lower urinary tract symptoms and erectile dysfunction.

Common Pitfalls in Some of the Experimental Studies in Erectile Function and Dysfunction: A Consensus Article

Experimental studies investigating physiology of erectile function and pathophysiology erectile dysfunction employ several in vitro and in vivo techniques. As the field of sexual medicine expanding, the proper conduct of such techniques is becoming an even more important necessity than before. This review article aims to guide scientists, particularly young researchers and new comers in the field, toward employment of these techniques in an appropriate, timely, and competent fashion. The authors reviewed the existing available published articles on the following topics: intracavernosal pressure measurements, cavernous nerve injury models, nitric oxide-cyclic guanosine monophosphate pathway, hypertension- and smoking-induced erectile dysfunction models, and stem cells. The authors present a consensus on how to best perform these models and techniques and also highlight the pitfalls. The authors hope that this article will assist and encourage young scientists in the field and that similar articles covering other important models will be also available to them soon.

Defective Nitric Oxide–Cyclic Guanosine Monophosphate Signaling in Patients With Bipolar Disorder

Bipolar disorder (BD) is associated with elevated cardiovascular mortality rates. We investigated the modulation of l-arginine-nitric oxide (NO) signaling in platelets from patients with BD at different phases. Platelets obtained from 28 patients with BD and 10 healthy volunteers were analyzed for l-arginine transport, NO synthase (NOS) activity, cyclic guanosine monophosphate content, and biomarkers of oxidative stress. Expressions of NOS isoforms, soluble guanylyl cyclase, and arginase were also measured in platelets. Amino acid and C-reactive protein levels in plasma were assessed. Plasma concentrations of l-arginine (mean [M] ± standard error of the mean [SEM] = 97 ± 10 versus 121 ± 10 µM) and its transport into platelets (median [interquartile range] = 26.0 [28.6] versus 26.5 [43.9] pmol/10(9) cells per minute) did not differ between patients with BD and controls (p > .05). Patients with BD showed reduced NOS activity (M ± SEM = 0.037 ± 0.003 versus 0.135 ± 0.022 pmol/10(8) cells, p < .001), but not endothelial NOS, inducible NOS, and arginase expression, compared with controls (p > .05). Cyclic guanosine monophosphate content was reduced (M ± SEM = 0.022 ± 0.003 versus 0.086 ± 0.020 pmol/10(8) cells, p < .05) despite the absence of changes in soluble guanylyl cyclase expression (median [interquartile range] = 21.6 [15.5] versus 9.5 [9.4] arbitrary units, p > .05) in patients with BD. Superoxide dismutase activity, but not catalase activity, was increased in patients with BD in the manic phase (M ± SEM = 2094 ± 335 versus 172 ± 17 U/mg protein, p < .001). C-reactive protein was elevated only in manic episodes (M ± SEM = 0.8 ± 0.2 versus 0.1 ± 0.02 mg/L, p < .001). Impaired NO generation from platelets, inflammation, and oxidative stress may play pivotal roles in the multifaceted process of cardiovascular events in BD.

VEGF genetic polymorphisms affect the responsiveness to sildenafil in clinical and postoperative erectile dysfunction

Vascular endothelial growth factor (VEGF) is a cytokine involved in angiogenesis and is closely related to the nitric oxide-cyclic guanosine monophosphate pathway, a target for sildenafil. We investigated for the first time whether three clinically relevant polymorphisms in the VEGF gene are associated with altered responsiveness to sildenafil treatment in postoperative erectile dysfunction (PED) and clinical erectile dysfunction (CED). We determined VEGF genotypes for three polymorphisms in VEGF promoter: -2578C>A (rs699947), -1154G>A (rs1570360) and -634G>C (rs2010963) in 126 patients with erectile dysfunction (ED; 66 patients with PED and 60 patients with CED). The patients were classified as good or poor responders to sildenafil (GR and PR groups, respectively) according to their responses with basis on the changes in five-item version of the International Index for Erectile Function (5-IIEF). We found an association of the -1154AA genotype with PR in both PED and CED patients (P<0.05), whereas the -2578AA and the -2578CA genotypes were associated with PR only in the CED group (P<0.05). The AAG haplotype was more common in PR than in GR patients (38% versus 20%, respectively; P=0.032) in the CED group, thus increasing the risk for a worse response to sildenafil (odds ratio, OR=2.33, 95% confidence interval, CI=1.07-5.09). However, this finding does not resist to Bonferroni's correction (P>0.0125). Our results indicate that VEGF polymorphisms affect the responsiveness of PED and CED patients to sildenafil. These findings may help to improve the therapy of patients with ED.

The role of the NMDA receptors and l-arginine–nitric oxide–cyclic guanosine monophosphate pathway in the antidepressant-like effect of duloxetine in the forced swimming test

Duloxetine is a selective serotonin and noradrenaline reuptake inhibitor used as antidepressant. However, its mechanisms of action are not fully understood. This study investigated the effect of duloxetine in the mouse forced swimming test (FST) and in the tail suspension test (TST) and the involvement of the NMDA receptors and the l-arginine–NO–cGMP pathway in its effect in the FST. Duloxetine reduced the immobility time both in the FST and in the TST (dose range of 1–30mg/kg, i.p.), without changing locomotion in an open-field. Duloxetine administered orally (1–30mg/kg) also reduced the immobility time in the FST. The effect of duloxetine (10mg/kg, p.o.) in the FST was prevented by pre-treatment with NMDA (0.1pmol/site, i.c.v.), d-serine (30μg/site, i.c.v.), (l-arginine (750mg/kg, i.p.), S-nitroso-N-acetyl-penicillamine (SNAP, 25μg/site, i.c.v) or sildenafil (5mg/kg, i.p.). The administration of MK-801 (0.001mg/kg, i.p.), 7-nitroindazole (50mg/kg, i.p.), methylene blue (20mg/kg, i.p.) or 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (30pmol/site i.c.v.) in combination with a sub-effective dose of duloxetine (0.3mg/kg, p.o.) reduced the immobility time in the FST. Moreover, the administration of duloxetine (10mg/kg) produced a reduction in NOx levels in the hippocampus and cerebral cortex. Altogether the results suggest that the effect of duloxetine in the FST is dependent on either a blockade of NMDA receptors or an inhibition of NO. In addition, our results further reinforce the role of NMDA receptors and l-arginine–NO–cGMP pathway, besides the monoaminergic systems, in the mechanism of action of current prescribed antidepressant agents.

Effects of Icariside II on Improving Erectile Function in Rats With Streptozotocin‐Induced Diabetes

Icariin and icariside II (ICA II), 2 active components isolated from herba epimedii, have a closely structural relationship. There is evidence that icariin may be useful in the treatment of erectile dysfunction (ED); however, the study on the therapeutic efficacy of ICA II on ED is currently scant. We investigated the effects of ICA II on improving erectile function of rats with streptozocin-induced diabetes. Fifty 8-week-old Sprague-Dawley rats were randomly distributed into normal control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozocin (60 mg/kg). Three days later, the diabetic rats were randomly divided into 4 groups including a saline-treated placebo arm and 3 ICA II-treated models (1, 5, and 10 mg/kg/d). After 3 months, penile hemodynamics was measured by cavernous nerve electrostimulation (CNE) with real time intracorporal pressure assessment. Penises were harvested with subsequent histological examination (picrosirius red stain, Hart elastin stain, and immunohistochemical stain) and Western blots to explore the expression of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and transforming growth factor β1 (TGFβ1)/Smad2 signaling pathways. Diabetes significantly attenuated erectile responses to CNE. Diabetic rats had decreased corpus cavernosum smooth muscle/collagen ratio and endothelial cell content relative to the control group. The ratio of collagen I to III was significantly lower in the corpora of diabetic rats; furthermore, cavernous elastic fibers were fragmented in the diabetic animals. Neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase, and vascular endothelial growth factor were expressed at lower levels in the diabetic group; ICA II-treated diabetic rats had higher expression in the penis relative to placebo-treated diabetic animals. Both the TGFβ1/Smad2/connective tissue growth factor (CTGF) signaling pathway and apoptosis were down-regulated in the penis from ICA II-treated rats. ICA II treatment attenuates diabetes-related impairment of penile hemodynamics, likely by increasing smooth muscle, endothelial function, and nNOS expression. ICA II could alter corpus cavernosum fibrous-muscular pathological structure in diabetic rats, which could be regulated by the TGFβ1/Smad2/CTGF and NO-cGMP signaling pathways.

Relaxin Signals through a RXFP1-pERK-nNOS-NO-cGMP-Dependent Pathway to Up-Regulate Matrix Metalloproteinases: The Additional Involvement of iNOS

The hormone, relaxin, inhibits aberrant myofibroblast differentiation and collagen deposition by disrupting the TGF-β1/Smad2 axis, via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), extracellular signal-regulated kinase (ERK)1/2 phosphorylation (pERK) and a neuronal nitric oxide (NO) synthase (nNOS)-NO-cyclic guanosine monophosphate (cGMP)-dependent pathway. However, the signalling pathways involved in its additional ability to increase matrix metalloproteinase (MMP) expression and activity remain unknown. This study investigated the extent to which the NO pathway was involved in human gene-2 (H2) relaxin's ability to positively regulate MMP-1 and its rodent orthologue, MMP-13, MMP-2 and MMP-9 (the main collagen-degrading MMPs) in TGF-β1-stimulated human dermal fibroblasts and primary renal myofibroblasts isolated from injured rats; by gelatin zymography (media) and Western blotting (cell layer). H2 relaxin (10–100 ng/ml) significantly increased MMP-1 (by ∼50%), MMP-2 (by ∼80%) and MMP-9 (by ∼80%) in TGF-β1-stimulated human dermal fibroblasts; and MMP-13 (by ∼90%), MMP-2 (by ∼130%) and MMP-9 (by ∼115%) in rat renal myofibroblasts (all p<0.01 vs untreated cells) over 72 hours. The relaxin-induced up-regulation of these MMPs, however, was significantly blocked by a non-selective NOS inhibitor (L-nitroarginine methyl ester (hydrochloride); L-NAME; 75–100 µM), and specific inhibitors to nNOS (N-propyl-L-arginine; NPLA; 0.2–2 µM), iNOS (1400W; 0.5–1 µM) and guanylyl cyclase (ODQ; 5 µM) (all p<0.05 vs H2 relaxin alone), but not eNOS (L-N-(1-iminoethyl)ornithine dihydrochloride; L-NIO; 0.5–5 µM). However, neither of these inhibitors affected basal MMP expression at the concentrations used. Furthermore, of the NOS isoforms expressed in renal myofibroblasts (nNOS and iNOS), H2 relaxin only stimulated nNOS expression, which in turn, was blocked by the ERK1/2 inhibitor (PD98059; 1 µM). These findings demonstrated that H2 relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to mediate its anti-fibrotic actions, and additionally signals through iNOS to up-regulate MMPs; the latter being suppressed by TGF-β1 in myofibroblasts, but released upon H2 relaxin-induced inhibition of the TGF-β1/Smad2 axis.

Tadalafil in pulmonary hypertension: May be more than seen?

I read an interesting article about the superiority of tadalafil over tempol via its antioxidant capacity on acute hypoxia-induced pulmonary hypertension (PH) model. Rashid et al. investigated and compared the antioxidant capacity of specific phosphodiesterase type-5 (PDE5) inhibitor, tadalafil, with superoxide dismutase mimetic and an efficient free radical scavenging agent tempol in acute hypoxia-induced PH model. They demonstrated that tadalafil was superior to tempol in inhibiting hypoxia-induced rise in right ventricular systolic pressure (RVSP) without producing any fall in mean arterial pressure. Also, tadalafil was shown to partially prevent oxidative stress. In conclusion, Rashid et al. highlighted the superiority of tadalafil especially in the settings of chronic hypoxia-induced PH. Monocrotaline (MCT)-induced PH is the other widely accepted animal model in PH studies. MCT is a member of the pyrrolizidine alkaloid family of plant toxins, which induces a delayed, yet progressive vascular injury resulting in PH in rats, dogs and monkeys. It is recognized that the initial reaction to MCT-induced PH is injury to the endothelial cells that precedes media hypertrophy in small size pulmonary arteries, and leads to an increase in pulmonary artery disease. These alterations in morphology largely mimic the primary PH in humans. We have recently compared the effects of three commercially available PDE5 inhibitors: sildenafil, vardenafil and tadalafil in rat MCT-induced PH model. It has been briefly demonstrated that vardenafil was more effective than sildenafil and tadalafil in this model. Vardenafil relaxes pulmonary artery rings in pulmonary hypertensive group through nitric oxide (NO)-cyclic guanosine monophosphate (NO-cGMP)-independent pathway. Neither sildenafil nor tadalafil achieves to induce relaxation response in pulmonary artery rings when NO-cGMP pathway inhibitor (NO synthase inhibitor, L-NAME and soluble guanylyl cyclase inhibitor, ODQ) incubations were used. This superiority potentially results from the differences in the heterocyclic ring system of vardenafil. Biochemical potencies (affinities) of these compounds for PDE5 have been determined by half maximal inhibitory concentration (IC50) and dissociation rate (KD), respectively, were the following: sildenafil (3.7 + 1.4, 3.7 + 0.29 nM), tadalafil (1.8 + 0.40, 1.9 + 0.37 nM), vardenafil (0.091 + 0.031, 0.27 + 0.01 nM), and it has been clearly demonstrated that vardenafil was the most potent inhibitor of PDE-5 because of its slow dissociation rate. Although tadalafil has an advantage over sildenafil and vardenafil because of its longer duration of action, tadalafil did not significantly alter the time to exercise treadmill test-induced ischemia compared with placebo in subjects with coronary artery disease. It is well-known that PH and coronary artery disease share similar risk factors, including endothelial dysfunction, diminishing of NO production and increased oxidative stress. This study is also in agreement with the others demonstrating that tadalafil was less effective than sildenafil and vardenafil in reducing the infarct size. In other preparations such as rat aorta-penile arteries and rabbit isolated pulmonary artery–corpus cavernosum tissue, vardenafil was shown to be more potent than other PDE5 inhibitors in relaxing the vascular tissues and preparations. These discrepancies may be attributed to experimental model, protocols or preparations difference employed in these studies. Also, these studies demonstrated that vardenafil has some beneficial effects in addition to its PDE5 inhibitory activity (e.g. activation of some ion channels or blocking of calcium influx).

Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle.

Acute atorvastatin treatment exerts antidepressant-like effect in mice via the l-arginine–nitric oxide–cyclic guanosine monophosphate pathway and increases BDNF levels

Atorvastatin is a synthetic and lipophilic statin that presents a good effect in decreasing cholesterol levels and is safe and well tolerated. Population-based studies have suggested a positive role of statins in reducing depression risk. This study aimed at investigating the atorvastatin effect in the tail suspension test (TST) and in the forced swimming test (FST). The participation of NMDA receptors and L-arginine-NO-cGMP in an atorvastatin antidepressant-like effect in the TST was evaluated. Acute atorvastatin administration (0.1-30 mg/kg) reduced the immobility time both in TST and FST. A similar effect was observed by using imipramine as a positive control in the TST and FST (1 and 0.1-1 mg/kg, p.o., respectively). An atorvastatin (0.1 mg/kg) antidepressant-like effect was prevented by the pretreatment of mice with NMDA (0.1 pmol/site, i.c.v.), L-arginine (750 mg/kg, i.p.) or sildenafil (5 mg/kg, i.p.). The administration of MK-801 (0.001 mg/kg, i.p.), ketamine (0.1 mg/kg, i.p.), 7-nitroindazole (50 mg/kg, i.p.), methylene blue (20 mg/kg, i.p.), or ODQ (30 pmol/site i.c.v.) in combination with a subeffective dose of atorvastatin (0.01 mg/kg, p.o.) reduced the immobility time in the TST compared to drugs alone, showing the participation of the pathway L-arginine-NO-cGMP. The administration of drugs did not produce any significant alteration in locomotor activity in the open-field test. Acute atorvastatin treatment (0.1-10.0 mg/kg, v.o.) increased the hippocampal BDNF levels, which is an effect that has not been observed in imipramine-treated mice. These results demonstrate that atorvastatin exerts an antidepressant-like effect and point to dependence on the inhibition of NMDA receptors and NO-cGMP synthesis, and on the increase of hippocampal BDNF levels.

Single atom substitution in mouse protein kinase G eliminates oxidant sensing to cause hypertension

Blood pressure regulation is crucial for the maintenance of health, and hypertension is a risk factor for myocardial infarction, heart failure, stroke and renal disease. Nitric oxide (NO) and prostacyclin trigger well-defined vasodilator pathways; however, substantial vasorelaxation in response to agents such as acetylcholine persists when the synthesis of these molecules is prevented. This remaining vasorelaxation activity, termed endothelium-derived hyperpolarizing factor (EDHF), is more prevalent in resistance than in conduit blood vessels and is considered a major mechanism for blood pressure control. Hydrogen peroxide (H2O2) has been shown to be a major component of EDHF in several vascular beds in multiple species, including in humans. H2O2 causes the formation of a disulfide bond between the two α subunits of protein kinase G I-α (PKGI-α), which activates the kinase independently of the NO-cyclic guanosine monophosphate (cGMP) pathway and is coupled to vasodilation. To test the importance of PKGI-α oxidation in the EDHF mechanism and blood pressure control in vivo, we generated a knock-in mouse expressing only a C42S 'redox-dead' version of PKGI-α. This amino acid substitution, a single-atom change (an oxygen atom replacing a sulfur atom), blocked the vasodilatory action of H2O2 on resistance vessels and resulted in hypertension in vivo.

Phosphodiesterase 5 Inhibition Attenuates Cerebral Vasospasm and Improves Functional Recovery After Experimental Subarachnoid Hemorrhage

Cerebral vasospasm is an independent predictor of poor outcome after subarachnoid hemorrhage (SAH). The nitric oxide-cyclic guanosine monophosphate (NO-cGMP) vasodilatory pathway is strongly implicated in its pathophysiology. Preliminary studies suggest that phosphodiesterase 5 (PDE5), an enzyme that degrades cGMP, may play a role because the PDE5 inhibitor sildenafil was found to reduce vasospasm after SAH. However, several questions that are critical when considering translational studies remain unanswered. To elucidate the mechanism of action of sildenafil against vasospasm and to assess whether sildenafil attenuates SAH-induced neuronal cell death, improves functional outcome after SAH, or causes significant physiological side effects when administered at therapeutically relevant doses. SAH was induced via endovascular perforation in male C57BL6 mice. Beginning 2 hours later, mice received sildenafil citrate (0.7, 2 or 5 mg/kg orally twice daily) or vehicle. Neurological outcome was assessed daily. Vasospasm was determined on post-SAH day 3. Brain PDE5 expression and activity, cGMP content, neuronal cell death, arterial blood pressure, and intracranial pressure were examined. We found that PDE5 activity (but not expression) is increased after SAH, leading to decreased cGMP levels. Sildenafil attenuates this increase in PDE5 activity and restores cGMP levels after SAH. Post-SAH initiation of sildenafil was found to decrease vasospasm and neuronal cell death and markedly improve neurological outcome without causing significant physiological side effects. Sildenafil, a US Food and Drug Administration-approved drug with a proven track record of safety in humans, is a promising new therapy for vasospasm and neurological deficits after SAH.

The Selective Phosphodiesterase 9 (PDE9) Inhibitor PF-04447943 Attenuates a Scopolamine-Induced Deficit in a Novel Rodent Attention Task

: Numerous changes occur during aging and Alzheimer's disease (AD) progression, including a decline in cholinergic functioning and cognition, as well as alterations in gene expression and activity in the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Donepezil, the current standard of care for Alzheimer's disease, improves cholinergic functioning and has demonstrated effects on multiple domains of cognition, including memory and attention in both preclinical species and patients. We previously found that increasing activation of the NO/cGMP pathway via phosphodiesterase 9 (PDE9) inhibition also improves memory in rodents and suggested that PDE9 might be a promising target for novel treatments for AD. Here we investigated whether PDE9 inhibition also enhances attention using a novel attention task in rats. We validated this task using several pharmacological manipulations and showed that the selective PDE9 inhibitor PF-04447943 produced effects similar to those of donepezil. These data confirm and extend the hypothesis that PDE9 inhibition might serve as a novel treatment for AD and age-related cognitive decline.

Hypoxic Regulation of Pulmonary Vascular Smooth Muscle Cyclic Guanosine Monophosphate–Dependent Kinase by the Ubiquitin Conjugating System

We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1α was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.

Effects of Icariin on Improving Erectile Function in Streptozotocin‐Induced Diabetic Rats

Icariin has been shown to improve penile hemodynamics in animal models of erectile dysfunction from cavernous nerve injury and castration. The effects of icariin on penile hemodynamics in diabetic animals remain to be determined. Transforming growth factor β1 (TGFβ1) has been implicated in the pathogenesis of diabetes-related erectile dysfunction. The aim of this study was to investigate the effects of icariin in the penis of streptozotocin (STZ)-induced diabetic rat. Two-month-old Sprague-Dawley male rats received one-time intraperitoneal (IP) STZ (60 mg/kg) or vehicle injection after a 16-hour fast. Three days later, the STZ-induced diabetic rats were randomly divided into four groups and were treated with daily gavage feedings of a 50:50 mix of normal saline and dimethyl sulfoxide (DMSO) or icariin dissolved in DMSO at doses of 1, 5, and 10 mg/kg for 3 months. A positive control group underwent IP injection of saline followed by daily gavage of saline/DMSO solution. Treatment was stopped 1 week prior to functional assay and euthanasia. Penile hemodynamics was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure (ICP) measurement. After euthanasia, penile tissue was studied using immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay (ELISA) to assess the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and TGFβ1/Smad2 signaling pathway. Diabetes attenuated ICP response in control animals. Untreated diabetic animals had decreased smooth muscle/collagen ratio and endothelial cell content in the corpora cavernosa; treatment with icariin partially attenuating these effects. Icariin-treated animals also had a significantly greater expression of nicotinamide adenine dinucleotide phosphate-positive nerves and the endothelial cell markers, von Willebrand factor (vWF), and platelet endothelial cell adhesion molecule-1 (PECAM). TGFβ1/Smad2 signaling pathway was down-regulated in the penis from icariin-treated models relative to what was observed in negative control animals. Icariin treatment preserved penile hemodynamics, smooth muscle and endothelial integrity, and neuronal nitric oxide synthase expression in the penis of diabetic rats. Down-regulation of TGFβ1/Smad2 signaling pathway might mediate this effect.