From single drug targets to synergistic network pharmacology in ischemic stroke.

Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein-protein interactions but also metabolite-dependent interactions. Based on this protein-metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4 Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood-brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein-metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

Neural regulation of the contractility of nutrient artery in the guinea pig tibia.

Nutrient arteries provide the endosteal blood supply to maintain bone remodelling and energy metabolism. Here, we investigated the distribution and function of perivascular nerves in regulating the contractility of the tibial nutrient artery. Changes in artery diameter were measured using a video tracking system, while the perivascular innervation was investigated using fluorescence immunohistochemistry. Nerve-evoked phasic constrictions of nutrient arteries were suppressed by phentolamine (1 µM), an α-adrenoceptor antagonist, guanethidine (10 µM), a blocker of sympathetic transmission, or fluoxetine (10 µM), a serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor. In arteries pretreated with guanethidine, residual nerve-evoked constrictions were abolished by a high concentration of propranolol (10 µM) that is known to inhibit 5-HT receptors, or ketanserin (100 nM), a 5-HT2 receptor antagonist, but not SB207216 (1 µM), an antagonist of 5-HT3 and 5-HT4 receptors. Bath-applied 5-HT (100 nM) induced arterial constriction that was suppressed by propranolol (10 µM) or ketanserin (100 nM). Nerve-evoked arterial constrictions were enhanced by spantide (1 µM), a substance P (SP) receptor antagonist, or L-nitro arginine (L-NA; 100 µM), an inhibitor of nitric oxide synthase (NOS). Immunohistochemistry revealed 5-HT-positive nerves running along the arteries that are distinct from perivascular sympathetic or substance P-positive primary afferent nerves. For the first time, functional serotonergic nerves are identified in the tibial nutrient artery of the guinea pig. Thus, it appears that tibial nutrient arterial calibre is regulated by the balance between sympathetic and serotonergic vasoconstrictor nerves and vasodilator afferent nerves that release substance P-stimulating endothelial nitric oxide (NO) release.

Guanosine, a guanine-based nucleoside relaxed isolated corpus cavernosum from mice through cGMP accumulation.

In corpus cavernosum (CC), guanosine triphosphate (GTP) is converted into cyclic guanosine monophosphate (cGMP) to induce erection. The action of cGMP is terminated by phosphodiesterases and efflux transporters, which pump cGMP out of the cell. The nucleotides, GTP, and cGMP were detected in the extracellular space, and their hydrolysis lead to the formation of intermediate products, among them guanosine. Therefore, our study aims to pharmacologically characterize the effect of guanosine in isolated CC from mice. The penis was isolated and functional and biochemical analyses were carried out. The guanine-based nucleotides GTP, guanosine diphosphate, guanosine monophosphate, and cGMP relaxed mice corpus cavernosum, but the relaxation (90.7 ± 12.5%) induced by guanosine (0.000001-1 mM) was greater than that of the nucleotides (~ 45%, P < 0.05). Guanosine-induced relaxation was not altered in the presence of adenosine type 2A and 2B receptor antagonists. No augment was observed in the intracellular levels of cyclic adenosine monophosphate in tissues stimulated with guanosine. Inhibitors of nitric oxide synthase (L-NAME, 100 µM) and soluble guanylate cyclase (ODQ, 10 µM) produced a significant reduction in guanosine-induced relaxation in all concentrations studied, while in the presence of tadalafil (300 nM), a significant increase was observed. Pre-incubation of guanosine (100 µM) produced a 6.6-leftward shift in tadalafil-induced relaxation. The intracellular levels of cGMP were greater when CC was stimulated with guanosine. Inhibitors of ecto-nucleotidases and xanthine oxidase did not interfere in the response induced by guanosine. In conclusion, our study shows that guanosine relaxes mice CC and opens the possibility to test its role in models of erectile dysfunction.

The effects of oral arginine on its metabolic pathways in Sprague-Dawley rats.

Oral arginine supplements are popular mainly for their presumed vasodilatory benefit. Arginine is a substrate for at least four enzymes including nitric oxide synthase (NOS) and arginase, but the impact of oral supplements on its different metabolic pathways is not clear. Deficiencies of arginine-metabolising enzymes are associated with conditions such as hyperammonaemia, endothelial dysfunction, central nervous system and muscle dysfunction, which complicate the use of oral arginine supplements. We examined the effect of l-arginine (l-Arg) and d-arginine (d-Arg), each at 500 mg/kg per d in drinking water administered for 4 weeks to separate groups of 9-week-old male Sprague-Dawley rats. We quantified the expression of enzymes and plasma, urine and organ levels of various metabolites of arginine. l-Arg significantly decreased cationic transporter-1 expression in the liver and the ileum and increased endothelial NOS expression in the aorta and the kidney and plasma nitrite levels, but did not affect the mean arterial pressure. l-Arg also decreased the expression of arginase II in the ileum, arginine:glycine amidinotransferase in the liver and the kidney and glyoxalase I in the liver, ileum and brain, but increased the expression of arginine decarboxylase and polyamines levels in the liver. d-Arg, the supposedly inert isomer, also unexpectedly affected the expression of some enzymes and metabolites. In conclusion, both l- and d-Arg significantly affected enzymes and metabolites in several pathways that use arginine as a substrate and further studies with different doses and treatment durations are planned to establish their safety or adverse effects to guide their use as oral supplements.

Jaburetox, a urease-derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea.

Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.

Skin blood flow response to topically applied methyl nicotinate: Possible mechanisms.

BACKGROUND: Methyl nicotinate (MN) induces a local cutaneous erythema in the skin and may be valuable as a local provocation in the assessment of microcirculation and skin viability. The mechanisms through which MN mediates its vascular effect are not fully known. The aim of this study was to characterize the vasodilatory effects of topically applied MN and to study the involvement of nitric oxide (NO), local sensory nerves, and prostaglandin-mediated pathways. METHODS: MN was applied on the skin of healthy subjects in which NO-mediated (L-NMMA), nerve-mediated (lidocaine/prilocaine), and cyclooxygenase-mediated (NSAID) pathways were selectively inhibited. Microvascular responses in the skin were measured using laser speckle contrast imaging (LSCI). RESULTS: NSAID reduced the MN-induced perfusion increase with 82% (P < .01), whereas lidocaine/prilocaine reduced it with 32% (P < .01). L-NMMA did not affect the microvascular response to MN. CONCLUSION: The prostaglandin pathway and local sensory nerves are involved in the vasodilatory actions of MN in the skin.

Mechanisms of sex differences in exercise capacity.

Sex differences are an important component of National Institutes of Health rigor. The goal of this investigation was to test the hypothesis that female mice have greater exercise capacity than male mice, and that it is due to estrogen, nitric oxide, and myosin heavy chain expression. Female C57BL6/J wild-type mice exhibited greater (P < 0.05) maximal exercise capacity for running distance (489 ± 15 m) than age-matched male counterparts (318 ± 15 m), as well as 20% greater work to exhaustion. When matched for weight or muscle mass, females still maintained greater exercise capacity than males. Increased type I and decreased type II myosin heavy chain fibers in the soleus muscle from females are consistent with fatigue resistance and better endurance in females compared with males. After ovariectomy, female mice no longer demonstrated enhanced exercise, and treatment of male mice with estrogen resulted in exercise capacity similar to that of intact females (485 ± 37 m). Nitric oxide synthase, a downstream target of estrogen, exhibited higher activity in female mice compared with male mice, P < 0.05, whereas ovariectomized females exhibited nitric oxide synthase levels similar to males. Nitric oxide synthase activity also increased in males treated with chronic estrogen to levels of intact females. Nitric oxide synthase blockade with Nω-nitro-l-arginine methyl ester eliminated the sex differences in exercise capacity. Thus estrogen, nitric oxide, and myosin heavy chain expression are important mechanisms mediating the enhanced exercise performance in females.

Striatal Protection in nNOS Knock-Out Mice After Quinolinic Acid-Induced Oxidative Damage.

Under pathological conditions, nitric oxide can become a mediator of oxidative cellular damage, generating an unbalance between oxidant and antioxidant systems. The participation of neuronal nitric oxide synthase (nNOS) in the neurodegeneration mechanism has been reported; the activation of N-methyl-D-aspartate (NMDA) receptors by agonist quinolinic acid (QUIN) triggers an increase in nNOS function and promotes oxidative stress. The aim of the present work was to elucidate the participation of nNOS in QUIN-induced oxidative stress in knock-out mice (nNOS-/-). To do so, we microinjected saline solution or QUIN in the striatum of wild-type (nNOS +/+), heterozygote (nNOS+/-), and knock-out (nNOS-/-) mice, and measured circling behavior, GABA content levels, oxidative stress, and NOS expression and activity. We found that the absence of nNOS provides a protection against striatal oxidative damage induced by QUIN, resulting in decreased circling behavior, oxidative stress, and a partial protection reflected in GABA depletion. We have shown that nNOS-derived NO is involved in neurological damage induced by oxidative stress in a QUIN-excitotoxic model.

The regulatory effect of flavonoids extracted from Abutilon theophrasti leaves on gene expression in LPS-induced ALI mice via the NF-κB and MAPK signaling pathways.

Context: ALI is a common disease characterized by acute pulmonary inflammatory disorder. Abutilon theophrasti Medik. (Malvaceae), as a Chinese traditional medicine, is used for the treatment of inflammation. Its main constituents are flavonoid compounds. Objective: This study investigates the regulatory effect of a TFE from Abutilon theophrasti leaves on gene expression in LPS-induced ALI mice via the NF-κB and MAPK signaling pathways. Materials and methods: Kunming mice were intragastrically administered TFE (0.25, 0.5, 1.0 g/kg) for 5 days, and then ALI was induced via intranasal administration 40 µg of LPS in 10 µL PBS after intragastric administration on the 5th day, and PBS and DEX (2 mg/kg) were negative and positive control groups, respectively. Results: The relative expression of iNOS gene was 0.707, 0.507 and 0.483 for 0.25, 0.5 and 1.0 g/kg TFE, and COX-2 gene expression was also reduced after treatment by three concentrations of TFE with 0.768, 0.545, and 0.478. The mRNA expression levels of p65 were 0.61, 0.43 and 0.27 for 0.25, 0.5 and 1.0 g/kg TFE and IκB levels were increased in a dose-dependent manner with 3.99, 13.69 and 34.36. 0.5 and 1.0 g/kg TFE inhibited the expression of ERK1/2 with 0.59 and 0.38, p38MAPK with 0.62 and 0.54, and JNK with 0.37 and 0.29, and JNK mRNA expression was 0.60 for 0.25 g/kg TFE. Discussion and conclusion: These results indicate that the regulatory mechanisms of TFE on gene expression in LPS-induced ALI mice include inhibition of the NF-κB and MAPK signaling pathways.

Modulation of murine intestinal immunity by Moringa oleifera extract in experimental hymenolepiasis nana.

The potential therapeutic value of Moringa oleifera extract (MOE), due to its anti-inflammatory and anti-oxidant effects, has been reported previously. In this study, Hymenolepis nana antigen (HNA) in combination with MOE was used in immunization against H. nana infection. Adult worm and egg counts were taken, while histological changes in the intestine were observed. Mucosal mast (MMCs) and goblet cells (GCs) were stained with specific stains, while serum and intestinal IgA were assayed using enzyme-linked immunosorbent assay (ELISA). Reduced glutathione (GSH) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) were assayed. Real-time polymerase chain reaction (PCR) was used for detection of mRNA expression in ileum tissue. The results demonstrated an improvement in the architecture of intestinal villi, decreased inducible nitric oxide synthase (iNOs) and TBARS, and increased GSH in HNA, MOE and MOE + HNA groups. In the same groups, an increase in GCs, mucin 2 (MUC2), interleukins (IL)-4, -5 and -9, and stem cell factor (SCF) versus a decrease in both interferon-gamma (IFN-γ) and transforming growth factor (TGF-ß) expression appeared. HNA and MOE + HNA increased serum and intestinal IgA, respectively. MOE decreased MMCs and achieved the highest reductions in both adult worms and eggs. In conclusion, MOE could achieve protection against H. nana infections through decreased TGF-ß, IFN-γ and MMC counts versus increased GC counts, T-helper cell type 2 (Th2) cytokines and IgA level.

[Effect of hydrogen sulfide on inducible nitric oxide synthase in kidneys of Type 1 diabetic rats].

OBJECTIVE: To investigate effects of hydrogen sulfide (H2S) on inducible nitric oxide synthase (iNOS) in kidneys of Type 1 diabetic rats.
 Methods: Thirty-two male SD rats were randomly divided into four groups: A normal control (NC) group, a diabetes mellitus (DM) group, a NaHS (NaHS+DM) group, and a NaHS control (NaHS) group (n=8 per group). Type 1 diabetes was induced by a single intraperitoneal injection of streptozotocin (55 mg/kg). After successful establishment of models, the rats in NaHS+DM and NaHS groups were injected with NaHS solution (56 µmol/kg) intraperitoneally. Eight weeks later, the activities of total nitric oxide synthase (T-NOS) and iNOS, as well as the level of nitric oxide (NO) were detected in serum and renal tissues, respectively. The activity of glutathione peroxidase (GSH-Px) was determined in renal tissues. The ultrastructures of renal tissues were observed by transmission electron microscope. The protein expression of iNOS in renal tissues was detected by Western blot.
 Results: Compared with the NC group, there was no significant difference in the various indexes in the NaHS group (P>0.05). However, in the DM group, the activities of T-NOS and iNOS, and the level of NO were all increased significantly in serum and renal tissues, while the activity of GSH-Px was decreased in renal tissues. Under the electronic microscope, the thickening of the glomerular capillary basement membrane, the proliferation of mesangial matrix, and the foot fusion were observed. The protein expression of iNOS was increased obviously in renal tissues in the DM group (P<0.01). Compared with the DM group, the activities of T-NOS and iNOS and the level of NO were all decreased in serum and renal tissues, while the activity of GSH-Px was increased in renal tissues in the NaHS+DM group (P<0.01). The renal ultrastructural damages were ameliorated obviously. The protein expression of iNOS was decreased significantly (P<0.01).
 Conclusion: H2S exerts a protective effect on kidney injury in type 1 diabetic rats. The mechanism might be related to inhibition of iNOS activity and protein expression, in turn leading to reduction of NO content in renal tissues.

[Effects of Ginaton on nitric oxide and nitric oxide synthase in patients with delayed encephalopathy after carbon monoxide poisoning].

Objective: To observe the effects of Ginaton on blood nitric oxide (NO) and nitric oxide synthase (NOS) in patients with delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Methods: A total of 116 patients with DEACMP who were treated in Emergency Department of Harrison International Peace Hospital Affiliated to Hebei Medical University from January 2012 to April 2016 were enrolled and ran-domly divided into control group and treatment group using a random number table, with 58 patients in each group. The patients in the control group were given conventional treatment including hyperbaric oxygen, preven-tion and treatment of cerebral edema, and promotion of brain cell metabolism, and those in the treatment group were given Ginaton in addition to the conventional treatment. The course of treatment was 2 weeks for both groups. The levels of neuron-specific enolase (NSE) , NO, NOS, and inducible nitric oxide synthase (iNOS) were measured before treatment and at 2 weeks after treatment, and the change in Mini-Mental State Examina-tion (MMSE) score and clinical outcome were observed in both groups. The correlation between the blood NO level on admission and the MMSE score was analyzed. Results: There was a significant difference in the overall response rate between the treatment group and the control group (81.03% vs 62.07%, χ(2) = 5.124, P=0.024). Be-fore treatment, there were no significant differences in the levels of NO and NSE, the activity of NOS and iN-OS, and MMSE score between the two groups (P>0.05). After treatment, both groups showed reductions in the levels of NO and NSE and the activity of NOS and iNOS, but the treatment group had significantly greater reduc-tions compared with the control group (P<0.05). Both groups showed a significant increase in the MMSE score after treatment, while the treatment group had a significantly greater increase compared with the control group (P<0.05). In the patients with DEACMP, the blood NO level on admission was negatively correlated with the MMSE score (r=-0.268, P=0.004). Conclusion: In the treatment of patients with DEACMP, Ginaton can effectively reduce the levels of NO and NSE and the activity of NOS and iNOS, increase the MMSE score, and promote the recovery of neurological function.

Insulin decreases atherosclerotic plaque burden and increases plaque stability via nitric oxide synthase in apolipoprotein E-null mice.

It has been argued whether insulin accelerates or prevents atherosclerosis. Although results from in vitro studies have been conflicting, recent in vivo mice studies demonstrated antiatherogenic effects of insulin. Insulin is a known activator of endothelial nitric oxide synthase (NOS), leading to increased production of NO, which has potent antiatherogenic effects. We aimed to examine the role of NOS in the protective effects of insulin against atherosclerosis. Male apolipoprotein E-null mice (8 wk old) fed a high-cholesterol diet (1.25% cholesterol) were assigned to the following 12-wk treatments: control, insulin (0.05 U/day via subcutaneous pellet), N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME, via drinking water at 100 mg/l), and insulin plus l-NAME. Insulin reduced atherosclerotic plaque burden in the descending aorta by 42% compared with control (plaque area/aorta lumen area: control, 16.5 ± 1.9%; insulin, 9.6 ± 1.3%, P < 0.05). Although insulin did not decrease plaque burden in the aortic sinus, macrophage accumulation in the plaque was decreased by insulin. Furthermore, insulin increased smooth muscle actin and collagen content and decreased plaque necrosis, consistent with increased plaque stability. In addition, insulin treatment increased plasma NO levels, decreased inducible NOS staining, and tended to increase phosphorylated vasodilator-stimulated phosphoprotein staining in the plaques of the aortic sinus. All these effects of insulin were abolished by coadministration of l-NAME, whereas l-NAME alone showed no effect. Insulin also tended to increase phosphorylated endothelial NOS and total neuronal NOS staining, effects not modified by l-NAME. In conclusion, we demonstrate that insulin treatment decreases atherosclerotic plaque burden and increases plaque stability through NOS-dependent mechanisms.

Etanercept improves endothelial function via pleiotropic effects in rat adjuvant-induced arthritis.

OBJECTIVES: To determine the effect of etanercept on endothelial dysfunction and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: At the first signs of arthritis, etanercept (10 mg/kg/3 days, s.c.) or saline was administered for 3 weeks in AIA rats. Body weights and arthritis scores were monitored daily. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase (COX-2), arginase, endothelium-derived hyperpolarizing factor and superoxide anions (O2 (-)°) production. Aortic expression of endothelial nitic oxide synthase (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22(phox) and p47(phox) was evaluated by western blotting analysis. Blood pressure, heart rate and blood levels of triglycerides, cholesterol and glucose were measured. RESULTS: Etanercept significantly reduced arthritis score (P < 0.001). It improved Ach-induced relaxation (P < 0.05) as a result of increased NOS activity, decreased COX-2/arginase activities and decreased O2 (-)° production. These functional effects relied on increased eNOS expression and phosphorylation, and decreased COX-2, arginase-2 and p22(phox) expressions. No correlation was found between arthritis score and Ach-induced relaxation. The treatment did not change triglycerides, cholesterol and glucose levels, but significantly increased systolic blood pressure and heart rate (P < 0.05). CONCLUSION: Our data demonstrated that efficient dosage of etanercept on inflammatory symptoms improved endothelial function in AIA. This beneficial effect on endothelial function is disconnected from its impact on CV risk factors and relates to pleiotropic effects of etanercept on endothelial pathways. These results suggest that etanercept could be a good choice for patients with rheumatoid arthritis at high risk of CV events.

Fenugreek seed extract attenuates cisplatin-induced testicular damage in Wistar rats.

Cisplatin (CIS) provides oxidative stress and inflammations in testicular tissues. Fenugreek seed extract (FSE) is a widely used herbal medicine with potent antioxidant and anti-inflammation properties. The purpose of this study was to investigate the protective effects and the possible mechanisms of FSE against CIS-induced testicular damage in rats. Adult male Wistar rats were given vehicle, single dose of CIS alone (10 mg kg(-1)), single dose of FSE alone or single dose of CIS followed by FSE (50, 100 or 200 mg kg(-1)) every day for 5 days. On day 6, oxidative stress and apoptotic testicular toxicity were evaluated. FSE attenuated both germ cell degenerations and apoptosis in seminiferous tubules in CIS-treated rats. Furthermore, FSE counteracted CIS-induced oxidative stress in rats as assessed by the restoration of superoxide dismutase and catalase activities and reduction in the myeloperoxidase activity and malondialdehyde levels in testes. CIS increased expressions of inducible nitric oxide synthase and nuclear factor-kappa B in testicular tissues. Importantly, treatment with FSE at all doses effectively alleviated all of these inflammatory parameters in testes. Based on these results, we concluded that FSE reduces CIS-induced reproductive toxicity in rats by the suppression of testicular oxidative stress, apoptosis and inflammations.

Ameliorative effects of pentoxifylline on NOS induced by diabetes in rat kidney.

OBJECTIVES: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide. The NO system has been implicated in the pathogenesis of DN. In this study, we aimed to evaluate the healing effect of pentoxifylline on NOS in STZ-induced diabetic rat's kidney. MATERIAL AND METHODS: In this study, 50 Wistar albino male rats were used. The rats were divided into five groups; Group C control; Group D only diabetes; Group D + PI and D + PII diabetes + pentoxifylline; Group P only pentoxifylline. Group DPI rats received just pentoxifylline from the beginning of the experiments. However, Group DPII rats received saline in the first month and 50 mg/kg/day of pentoxifylline for the following month. At the end of two months, NOS expressions in kidney tissue were assessed using qRT-PCR and immunohistochemistry analysis. RESULTS: At the end of the experiments, desquamation of the epithelial cells of the tubules, clear glycogen-filled distal tubules and increased number of apoptotic cells were seen in Group D. Diabetic rats' nNOS immunoreactivity had increased and eNOS and iNOS immunoreactivity had decreased; nNOS, iNOS and eNOS mRNA levels tended to decrease compared to the control group. PTX ameliorated eNOS, iNOS and nNOS protein levels and apoptotic cells, but did not affect mRNA levels. CONCLUSION: In conclusion, PTX has a healing effect on this damage by affecting NOS expression.

Sepiapterin prevents left ventricular hypertrophy and dilatory remodeling induced by pressure overload in rats.

Uncoupling of nitric oxide (NO) synthase (NOS) has been implicated in left ventricular (LV) hypertrophy (LVH) and dilatory remodeling induced by pressure overload. We investigated whether administration of sepiapterin, a substrate of the salvage pathway of tetrahydrobiopterin synthesis, prevents LVH and dilatory LV remodeling by inhibiting NOS uncoupling and increasing bioavailable NO. Pressure overload was induced in rats by transverse aortic constriction (TAC). Concentric LVH developed during 8 wk after TAC, and dilatory LV remodeling and dysfunction developed between 8 and 16 wk after TAC associated with a decrease in capillary density. Oral administration of sepiapterin or the superoxide/peroxynitrite scavenger N-(2-mercaptopropionyl)-glycine for 8 wk after TAC inhibited oxidative stress, but only sepiapterin increased bioavailable NO and inhibited cardiomyocyte hypertrophy associated with a further increase in capillary density. When sepiapterin was administered between 8 and 16 wk after TAC, cardiomyocyte hypertrophy was regressed and capillary density was restored. This was associated with the inhibition of interstitial fibrosis and dilatory LV remodeling. N-nitro-l-arginine methyl ester abrogated all the beneficial effects of sepiapterin in rats with TAC. These results suggest that sepiapterin prevents concentric LVH and dilatory remodeling after TAC primarily by increasing the bioavailability of NO.

Effects of melatonin on the nitric oxide system and protein nitration in the hypobaric hypoxic rat hippocampus.

BACKGROUND: It is well documented that the nitric oxide (NO) might be directly involved in brain response to hypobaric hypoxia, and could contribute to memory deficiencies. Recent studies have shown that melatonin could attenuate hypoxia or ischemia-induced nerve injuries by decreasing the production of free radicals. The present study, using immunohistochemical and immunoblot methods, aimed to explore whether melatonin treatment may affect the expression of nitric oxide system and protein nitration, and provide neuroprotection in the rat hippocampus injured by hypobaric hypoxia. Prior to hypoxic treatment, adult rats were pretreated with melatonin (100 mg/kg, i.p.) before they were exposed to the altitude chamber with 48 Torr of the partial oxygen concentration (pO2) for 7 h to mimic the ambience of being at 9000 m in height. They were then sacrificed after 0 h, 1, and 3 days of reoxygenation. RESULTS: The results obtained from the immunohistochemical and immunoblotting analyses showed that the expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine (Ntyr) and Caspase 3 in the hypoxic hippocampus were increased from 0 h to 3 days of reoxygenation. Interestingly, the hypoxia-induced increase of nNOS, eNOS, iNOS, Ntyr and Caspase 3 protein expression was significantly depressed in the hypoxic rats treated with melatonin. CONCLUSIONS: Activation of the nitric oxide system and protein nitration constitutes a hippocampal response to hypobaric hypoxia and administration of melatonin could provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaric hypoxia.

Tetrahydrobiopterin lowers muscle sympathetic nerve activity and improves augmentation index in patients with chronic kidney disease.

Chronic kidney disease (CKD) is characterized by overactivation of the sympathetic nervous system (SNS) that contributes to cardiovascular risk. Decreased nitric oxide (NO) bioavailability is a major factor contributing to SNS overactivity in CKD, since reduced neuronal NO leads to increased central SNS activity. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase that increases NO bioavailability in experimental models of CKD. We conducted a randomized, double-blinded, placebo-controlled trial testing the benefits of oral sapropterin dihydrochloride (6R-BH4, a synthetic form of BH4) in CKD. 36 patients with CKD and hypertension were randomized to 12 wk of 1) 200 mg 6R-BH4 twice daily + 1 mg folic acid once daily; vs. 2) placebo + folic acid. The primary endpoint was a change in resting muscle sympathetic nerve activity (MSNA). Secondary endpoints included arterial stiffness using pulse wave velocity (PWV) and augmentation index (AIx), endothelial function using brachial artery flow-mediated dilation and endothelial progenitor cells, endothelium-independent vasodilatation (EID), microalbuminuria, and blood pressure. We observed a significant reduction in MSNA after 12 wk of 6R-BH4 (-7.5 ± 2.1 bursts/min vs. +3.2 ± 1.3 bursts/min; P = 0.003). We also observed a significant improvement in AIx (by -5.8 ± 2.0% vs. +1.8 ± 1.7 in the placebo group, P = 0.007). EID increased significantly (by +2.0 ± 0.59%; P = 0.004) in the 6R-BH4 group, but there was no change in endothelial function. There was a trend toward a reduction in diastolic blood pressure by -4 ± 3 mmHg at 12 wk with 6R-BH4 (P = 0.055). 6R-BH4 treatment may have beneficial effects on SNS activity and central pulse wave reflections in hypertensive patients with CKD.

Tapentadol and nitric oxide synthase systems.

Tapentadol, a new analgesic drug with a dual mechanism of action (µ-opioid receptor agonism and norepinephrine reuptake inhibition), is indicated for the treatment of moderate to severe acute and chronic pain. In this paper, the possible additional involvement of the nitric oxide synthase (NOS) system in the antinociceptive activity of tapentadol was investigated using an unspecific inhibitor of NOS, L-NOArg, a relatively specific inhibitor of neuronal NOS, 7-NI, a relatively selective inhibitor of inducible NOS, L-NIL, and a potent inhibitor of endothelial NOS, L-NIO. Tapentadol (1-10 mg/kg, intraperitoneal) increased the threshold for mechanical (Randall-Selitto test) and thermal (tail-flick test) nociceptive stimuli in a dose-dependent manner. All four NOS inhibitors, administered intraperitoneally in the dose range 0.1-10 mg/kg, potentiated the analgesic action of tapentadol at a low dose of 2 mg/kg in both models of pain. We conclude that NOS systems participate in tapentadol analgesia.