Guanosine Monophosphate Levels Research Articles

Control of Orthodontic Tooth Movement by Nitric Oxide Releasing Nanoparticles in Sprague-Dawley Rats.

Orthodontic treatment commonly requires the need to prevent movement of some teeth while maximizing movement of other teeth. This study aimed to investigate the influence of locally injected nitric oxide (NO) releasing nanoparticles on orthodontic tooth movement in rats.Materials and Methods:Experimental tooth movement was achieved with nickel-titanium alloy springs ligated between the maxillary first molar and ipsilateral incisor. 2.2 mg/kg of silica nanoparticles containing S-nitrosothiol groups were injected into the mucosa just mesial to 1st molar teeth immediately prior to orthodontic appliance activation. NO release from nanoparticles was measured in vitro by chemiluminescence. Tooth movement was measured using polyvinyl siloxane impressions. Bones were analyzed by microcomputed tomography. Local tissue was assessed by histomorphometry.Results:Nanoparticles released a burst of NO within the first hours at approximately 10 ppb/mg particles that diminished by 10 × to approximately 1 ppb/mg particles over the next 1–4 days, and then diminished again by tenfold from day 4 to day 7, at which point it was no longer measurable. Molar but not incisor tooth movement was inhibited over 50% by injection of the NO releasing nanoparticles. Inhibition of molar tooth movement occurred only during active NO release from nanoparticles, which lasted for approximately 1 week. Molar tooth movement returned to control levels of tooth movement after end of NO release. Alveolar and long bones were not impacted by injection of the NO releasing nanoparticles, and serum cyclic guanosine monophosphate (cGMP) levels were not increased in animals that received the NO releasing nanoparticles. Root resorption was decreased and periodontal blood vessel numbers were increased in animals with appliances that were injected with the NO releasing nanoparticles as compared to animals with appliances that did not receive injections with the nanoparticles.Conclusion:Nitric oxide (NO) release from S-nitrosothiol containing nanoparticles inhibits movement of teeth adjacent to the site of nanoparticle injection for 1 week. Additional studies are needed to establish biologic mechanisms, optimize efficacy and increase longevity of this orthodontic anchorage effect.

Long-Term PDE-5A Inhibition Improves Myofilament Function in Left and Right Ventricular Cardiomyocytes through Partially Different Mechanisms in Diabetic Rat Hearts.

Heart failure with preserved ejection fraction (HFpEF) and right ventricular (RV) dysfunction are frequent complications of diabetic cardiomyopathy. Here we aimed to characterize RV and left ventricular (LV) remodeling and its prevention by vardenafil (a long-acting phosphodiesterase-5A (PDE-5A) inhibitor) administration in a diabetic HFpEF model. Zucker Diabetic Fatty (ZDF) and control, ZDF Lean (Lean) male rats received 10 mg/kg vardenafil (ZDF + Vard; Lean + Vard) per os, on a daily basis for a period of 25 weeks. In vitro force measurements, biochemical and histochemical assays were employed to assess cardiomyocyte function and signaling. Vardenafil treatment increased cyclic guanosine monophosphate (cGMP) levels and decreased 3-nitrotyrosine (3-NT) levels in the left and right ventricles of ZDF animals, but not in Lean animals. Cardiomyocyte passive tension (Fpassive) was higher in LV and RV cardiomyocytes of ZDF rats than in those receiving preventive vardenafil treatment. Levels of overall titin phosphorylation did not differ in the four experimental groups. Maximal Ca2+-activated force (Fmax) of LV and RV cardiomyocytes were preserved in ZDF animals. Ca2+-sensitivity of isometric force production (pCa50) was significantly higher in LV (but not in RV) cardiomyocytes of ZDF rats than in their counterparts in the Lean or Lean + Vard groups. In accordance, the phosphorylation levels of cardiac troponin I (cTnI) and myosin binding protein-C (cMyBP-C) were lower in LV (but not in RV) cardiomyocytes of ZDF animals than in their counterparts of the Lean or Lean + Vard groups. Vardenafil treatment normalized pCa50 values in LV cardiomyocytes, and it decreased pCa50 below control levels in RV cardiomyocytes in the ZDF + Vard group. Our data illustrate partially overlapping myofilament protein alterations for LV and RV cardiomyocytes in diabetic rat hearts upon long-term PDE-5A inhibition. While uniform patterns in cGMP, 3-NT and Fpassive levels predict identical effects of vardenafil therapy for the diastolic function in both ventricles, the uneven cTnI, cMyBP-C phosphorylation levels and pCa50 values implicate different responses for the systolic function.

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362−, and CD362+ MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model.Materials: For this purpose, 1 × 106 WT, CD362−, or CD362+ MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Leprdb/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis.Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362−, and CD362+ MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362+ MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362− MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3+ and CD68+ cells in db/db mice, whereas only splenocytes from CD362−- and CD362+-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice.Conclusion: CD362+ MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362− MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362+ MSC application was insufficient to improve diastolic function.

Efficacy of the Piperidine Nitroxide 4-MethoxyTEMPO in Ameliorating Serum Amyloid A-Mediated Vascular Inflammation

Intracellular redox imbalance in endothelial cells (EC) can lead to endothelial dysfunction, which underpins cardiovascular diseases (CVD). The acute phase serum amyloid A (SAA) elicits inflammation through stimulating production of reactive oxygen species (ROS). The cyclic nitroxide 4-MethoxyTEMPO (4-MetT) is a superoxide dismutase mimetic that suppresses oxidant formation and inflammation. The aim of this study was to investigate whether 4-MetT inhibits SAA-mediated activation of cultured primary human aortic EC (HAEC). Co-incubating cells with 4-MetT inhibited SAA-mediated increases in adhesion molecules (VCAM-1, ICAM-1, E-selectin, and JAM-C). Pre-treatment of cells with 4-MetT mitigated SAA-mediated increases in transcriptionally activated NF-κB-p65 and P120 Catenin (a stabilizer of Cadherin expression). Mitochondrial respiration and ROS generation (mtROS) were adversely affected by SAA with decreased respiratory reserve capacity, elevated maximal respiration and proton leakage all characteristic of SAA-treated HAEC. This altered respiration manifested as a loss of mitochondrial membrane potential (confirmed by a decrease in TMRM fluorescence), and increased mtROS production as assessed with MitoSox Red. These SAA-linked impacts on mitochondria were mitigated by 4-MetT resulting in restoration of HAEC nitric oxide bioavailability as confirmed by assessing cyclic guanosine monophosphate (cGMP) levels. Thus, 4-MetT ameliorates SAA-mediated endothelial dysfunction through normalising EC redox homeostasis. Subject to further validation in in vivo settings; these outcomes suggest its potential as a therapeutic in the setting of cardiovascular pathologies where elevated SAA and endothelial dysfunction is linked to enhanced CVD.

Prokaryotic Expression, Purification and Activity Detection of Phosphodiesterase 2A Protein

Depression is associated with changes in cyclic guanosine monophosphate (cGMP) levels. Depression can be improved by increasing the cGMP concentration through the cGMP/PKG pathway with PDE2A inhibitors. This study is aimed to improve the expression of a highly active PDE2A protein with an Escherichia coli vector ST6 for the screening of PDE2A inhibitors. PDE2A gene was obtained through polymerase chain reaction. A recombinant plasmid of ST6-PDE2A was built by seamless cloning and then introduced into E. coli BL21 (DE3). The cultivation conditions were optimized to increase target protein expression. The expressed protein was purified with Ni-NTA affinity chromatography. Its purified activity was measured by a PDE-GloTM phosphodiesterase kit. An maximized protein expression was obtained by cultivating E. coli BL21 with ST6-PDE2A in the YT medium at 37 °C till OD600 reached to 0.6-0.8 and then by inducible expressing with 1 mM IPTG at 16 °C for 40 hours. The resultant active protein has an EC50 of 0.1196 mg/ml.

A Selective Phosphodiesterase 10A Inhibitor Reduces L-Dopa-Induced Dyskinesias in Parkinsonian Monkeys.

Phosphodiesterase 10A is a member of the phosphodiesterase family whose brain expression is restricted to the striatum. Phosphodiesterase 10A regulates cyclic adenosine monophosphate and cyclic guanosine monophosphate, which mediate responses to dopamine receptor activation, and the levels of these cyclic nucleotides are decreased in experimental models of l-dopa-induced dyskinesia. The elevation of cyclic adenosine monophosphate/cyclic guanosine monophosphate levels by phosphodiesterase 10A inhibition may thus be targeted to reduce l-dopa-induced dyskinesia. The present study was aimed at determining the potential antidyskinetic effects of phosphodiesterase 10A inhibitors in a primate model of Parkinson's disease (PD). The experiments performed in this model were also intended to provide translational data for the design of future clinical trials. Five MPTP-treated macaques with advanced parkinsonism and reproducible l-dopa-induced dyskinesia were used. MR1916, a selective phosphodiesterase 10A inhibitor, at doses 0.0015 to 0.05 mg/kg, subcutaneously, or its vehicle (control test) was coadministered with l-dopa methyl ester acutely (predetermined optimal and suboptimal subcutaneous doses) and oral l-dopa chronically as daily treatment for 5 weeks. Standardized scales were used to assess motor disability and l-dopa-induced dyskinesia by blinded examiners. Pharmacokinetics was also examined. MR1916 consistently reduced l-dopa-induced dyskinesia in acute tests of l-dopa optimal and suboptimal doses. Significant effects were present with every MR1916 dose tested, but the most effective was 0.015 mg/kg. None of the MR1916 doses tested affected the antiparkinsonian action of l-dopa at the optimal dose. The anti-l-dopa-induced dyskinesia effect of MR1916 (0.015 mg/kg, subcutaneously) was sustained with chronic administration, indicating that tolerance did not develop over the 5-week treatment. No adverse effects were observed after MR1916 administration acutely or chronically. Results show that regulation of striatal cyclic nucleotides by phosphodiesterase 10A inhibition could be a useful therapeutic approach for l-dopa-induced dyskinesia, and therefore data support further studies of selective phosphodiesterase 10A inhibitors for PD therapy. © 2018 International Parkinson and Movement Disorder Society.

‘Have you had Viagra within the past 24 hours?’

Rapid drug-induced vasodilation is essential in treating coronary medical emergencies. Through oral or sublingual administration of glyceryl trinitrate (GTN), changes occur in cyclic guanosine monophosphate (cGMP) levels, as well as a reduction in blood pressure. Patients who require GTN treatment may also take vasodilatory medication for other medical conditions, including erectile dysfunction (ED). Current guidance in the UK contraindicates GTN use alongside sildenafil (commercially known as Viagra) and other related drugs, owing to the likelihood of life-threatening hypotension. Therefore, it is essential that paramedics and emergency responders establish whether patients have taken sildenafil within the past 24 hours before administering GTN. This article identifies the action mechanisms of both drugs, and provides an evidence-based explanation for co-administration outcomes; a dramatic drop in blood pressure which can potentially be fatal. This article also reflects on the challenges faced by paramedics when considering administration of GTN to determine whether sildenafil has been taken within the past 24 hours.

Effect of incubation with L-citrulline ex vivo on relaxation dysfunction of corpus cavernosum from type 2 diabetic rats

Objective To explore the role of the changes in endogenous L-citrulline levels and arginases expression in erectile dysfunction of diabetic rats, and investigate the effect of L-citrulline incubation ex vivo on the erectile dysfunction. Methods Type 2 diabetic rats were induced by high fat diet plus a single intraperitoneal injection of small dose streptozotocin (30 mg/kg), followed by high fat diet for 8 weeks. The erectile function of rats was reflected by the relaxation response to acetylcholine (ACh) of corpus cavernosum detected by isometric tension measurement. Levels of endogenous nitric oxide inhibitor asymmetric dimethylarginine (ADMA) and cyclic guanosine monophosphate (cGMP) were measured by enzyme-linked immunosorbent assay (ELISA). The content of L-citrulline, nitric oxide (NO) and the activity of nitric oxide synthase (NOS) were determined with colorimetric method. Western blotting was applied to detect the protein expression of NOS, arginases and phosphodiesterase 5 (PED5). Results The relaxation response to ACh of corpus cavernosum from diabetic rats was significantly lower than that of control rats (P<0.01), indicating the erectile dysfunction of diabetic rats. This erectile dysfunction was companied by the decreases of L-citrulline levels in serum and corpus cavernosum as well as up-regulation of arginaseⅠ& Ⅱ expression in the corpus cavernosum of diabetic rats. Incubation with L-citrulline (3 mmol/L) for 45 min ex vivo im-proved the relaxation function of corpus cavernosum from diabetic rats (P<0.05), while incubation with PED5 inhibitor silaenafil (3 μmol/L) had no significant effect. Furthermore, the ADMA level was increased, while the NOS activity, cGMP and NO contents were decreased in corpus cavernosum of diabetic rats compared to control rats. Down-regulation of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein expression as well as up-regulation of inducible nitric oxide synthase (iNOS) and PDE5 protein expression were also observed in the corpus cavernosum of diabetic rats. Conclusions The decreased endogenous L-citrulline levels and upregulated arginases expression was closely related to erectile dysfunction of diabetic rats, and the mechanism is associated to the up-regulation of arginase activity and decrease of NO synthesis in corpus cavernosum. L-citrulline incubation ex vivo exerted therapeutic effect on the relaxation dysfunction of corpus cavernosum from diabetic rats. Key words: L-citrulline; Diabetes mellitus; Erectile dysfunction; Arginase; Nitric oxide

LOW URINARY CGMP/BNP RATIO IS ASSOCIATED WITH WORSE OUTCOMES IN HEART FAILURE BUT IS INCREASED BY TREATMENT WITH SACUBITRIL/VALSARTAN: AN ANALYSIS OF PARADIGM-HF

Background: Urinary cyclic guanosine monophosphate (ucGMP) levels reflect the activity of vasoactive substances (eg B-type natriuretic peptide (BNP)). The ucGMP/BNP ratio is lower in heart failure (HF) and is associated with worse prognosis. We examined the relationship between ucGMP/BNP and

The effect of red ginseng and ginseng leaves on the substance and energy metabolism in hypothyroidism rats

BackgroundRecent studies have revealed that the properties Traditional Chinese Medicine is mostly associated with are substance and energy metabolism. Our study aimed to compare the effect of red ginseng (RG) (warm property) and ginseng leaves (GL; cold property) on the substance and energy metabolism of rats with hypothyroidism. Materials and methodsRats were administered propylthiouracil intraperitoneally for 20 d to cause hypothyroidism. The reference group was orally administered Aconiti Lateralis Radix Praeparaia [FZ (Fuzi in Chinese)], while both the RG and GL groups were orally administrated crude drugs. The rectal, tail, toe, and axilla temperature of the rats were assayed every 3 d. Oxygen consumption, carbon dioxide production, heat production, and energy expenditure were measured via TSE phenoMaster/LabMaster animal monitoring system. Adenosine monophosphate-activated protein kinase, Na+-K+-ATPase, fumarase, pyruvic acid and cyclic adenosine monophosphate/cyclic guanosine monophosphate were determined. ResultsThe lower levels of triiodothyronine, tetraiodothyronine, and thyrotropin-releasing hormone and the higher level of thyroid stimulating hormone revealed the successful establishment of a hypothyroidism model. Oxygen consumption, carbon dioxide production, heat production, and energy expenditure in the FZ and RG groups were obviously increased. The activity of Na+-K+-ATPase and fumarase in the FZ and RG groups was significantly increased. The cyclic adenosine monophosphate/cyclic guanosine monophosphate level in the FZ and RG groups was increased, while the GL group showed the opposite. ConclusionOur research provides a new way to explore the efficiency of Chinese medicine on the basis of the relationship between drug property and effects on substance and energy metabolism.

N-methylnicotinamide protects against endothelial dysfunction and attenuates atherogenesis in apolipoprotein E-deficient mice.

Epidemiological studies have demonstrated that N-methylnicotinamide (MNA) may exert antithrombotic and anti-inflammatory effects on the endothelium. However, the exact role of MNA in endothelial function remains uncertain. Apolipoprotein E-deficient (apoE(-/-) ) mice fed with a high-fat, high-cholesterol diet (HCD) and human umbilical vein endothelial cells (HUVECs) were used to explore the role of MNA in endothelial function and its underlying mechanism. The endothelium-dependent vasorelaxation to acetylcholine in the aortas of low and high dose MNA-fed apoE(-/-) mice was improved by 24 and 36% (p < 0.05), respectively, compared with high-fat, HCD-fed control. MNA significantly increased nitric oxide/cyclic guanosinemonophosphate levels and decreased asymmetric dimethylarginine (ADMA) concentrations by induction of dimethylarginine dimethylaminohydrolase (DDAH)2 both in aorta and endothelial cells. Neither the activity nor the protein expression of DDAH1 was influenced upon MNA treatment. Then, DDAH2 depletion by RNA interference in HUVECs abolished the protective effect of MNA on endothelial function. Mechanically, this could be attributed to a direct modulation of the methylation level of DDAH2 gene promoter region by MNA. The present study reveals a novel mechanism through which MNA improves endothelial dysfunction and attenuates atherogenesis via the modulation of ADMA-DDAH axis.

Riociguat and sildenafil for pulmonary hypertension: similarity and difference

Last decades, several drugs for treatment of pulmonary hypertension have been implemented in clinical practice. Sildenafil selectively inhibits phosphodiesterase-5 (PDE-5), increases cyclic guanosine monophosphate (cGMP) level in the pulmonary vascular wall leading to vasodilation, inhibition of proliferation and other specific effects. Sildenafil is indicated as single therapy and in sequential combination therapy of pulmonary arterial hypertension (PAH) (high-level evidence, high-class recommendation). However, therapeutic response on PDE-5 inhibitors is insufficient in a certain number of patients. Soluble guanylate cyclase (sGC) activators were proposed as a good alternative for such patients. Riociguat has dual mechanism of action: firstly, it is designed to increase cGMP biosynthesis via direct activation of sGC; this pathway resembles that of nitric oxide (NO) but is independent on NO. Secondly, riociguat can sensitise sGC to NO. Riociguat is indicated as single therapy and in sequential combination therapy of PAH and to treatment of chronic thromboembolic pulmonary hypertension (high-level evidence, high-class recommendation). A clinical trial RESPITE has been initiated in February, 2014, which is intended to evaluate efficacy of switching PAH patients from PDE-5 inhibitors to riociguat. Potential advantage of this drug is the mechanism independent of NO and of other PDE enzyme isoforms. Whether this advantage is clinically significant, remains to be seen, probably, in the RESPITE trial.

Uncoupling of endothelial nitric oxide synthase in cerebral vasculature of Tg2576 mice.

In this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω-nitro-L-arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator-activated receptor--delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5-cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub-optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimer's disease. Existing evidence suggests that Aβ peptides-induced up-regulation of expression and activity of NADPH oxidase causes increased production of superoxide anion (.O2(-)). .O2(-) can also be converted to hydrogen peroxide (H2O2) by enzymatic activity of superoxide dismutase (SOD) or spontaneous dismutation. Elevation of .O2(-) and H2O2 might cause oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and subsequent uncoupling of endothelial nitric oxide synthase (eNOS) (a) thus reducing levels of nitric oxide (NO) and 3',5'-cyclic guanosine monophosphate (cGMP). Supplementation of BH4 or activation of PPARδ prevents detrimental effects of eNOS uncoupling by restoring bioavailability of BH4 and scavenging of .O2(-), respectively (b). Activation of PPARδ also increases expression of catalase thereby inactivating H2O2. Generation of H2O2 by uncoupled eNOS in cerebral microvessels of Tg2576 mice is hypothetical.

Notoginsenoside Ft1 activates both glucocorticoid and estrogen receptors to induce endothelium-dependent, nitric oxide-mediated relaxations in rat mesenteric arteries

Panax notoginseng (Burk.) F.H. Chen has been used traditionally for the treatment of cardiovascular diseases. Notoginsenoside Ft1 (Ft1) is a bioactive saponin from the leaves of P. notoginseng. Experiments were designed to determine whether or not Ft1 is an endothelium-dependent vasodilator. Rat mesenteric arteries were suspended in organ chambers for the measurement of isometric tension during phenylephrine-induced contractions. The cyclic guanosine monophosphate (cGMP) level was assessed using enzyme immunoassay. The phosphorylation and protein expressions of endothelial nitric oxide synthase (eNOS), glucocorticoid receptors (GR), estrogen receptors beta (ERß), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) were determined by Western blotting. The localization of GR and ERß were determined by immunofluorescence staining. Ft1 caused endothelium-dependent relaxations, which were abolished by l-NAME (inhibitor of nitric oxide synthases) and ODQ (inhibitor of soluble guanylyl cyclase). Ft1 increased the cGMP level in rat mesenteric arteries. GR and ERß were present in the endothelial layer and their antagonism by RU486 and PHTPP, respectively, inhibited Ft1-induced endothelium-dependent relaxations and phosphorylations of eNOS, Akt and ERK1/2. Inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin and ERK1/2 by U0126 reduced Ft1-evoked relaxations and eNOS phosphorylation. Taken in conjunction, the present findings suggest that Ft1 stimulates endothelial GRs and ERßs with subsequent activation of the PI3K/Akt and ERK1/2 pathways in rat mesenteric arteries. This results in phosphorylation of eNOS and the release of NO, which activates soluble guanylyl cyclase in the vascular smooth muscle cells leading to relaxations.

Influence of Nitric Oxide and cGMP on Agonist-Induced Platelet Adhesion – An In Vitro Study in Platelets Isolated from Patients with Liver Cirrhosis / Uticaj Azot-Oksida I cGMP Na Adheziju Trombocita Izazvanu Agonistima – In Vitro Studija Na Izolovanim Trombocitima Pacijenata Sa Cirozom Jetre

Summary BackgroundVariceal bleeding, one of the major complications of liver cirrhosis, is primarily due to platelet activation defect and secondarily due to coagulation defects. Platelet adhesion is the key event in hemostasis. Since nitric oxide (NO) related stress is known to influence platelet functions in liver cirrhosis, we undertook the present study to evaluate the possible mechanism involved in the inhibition of platelet adhesion by NO.Methods:Agonist-induced platelet adhesion in vitro was measured in platelets isolated from normal subjects and cirrhosis patients. The time-dependent changes in nitric oxide synthase (NOS), NO, 3',5'-cyclic guanosine monophosphate (cGMP) and cytosolic calcium (Ca2+) levels were monitored during adhesion. The percentage of platelet adhesion was also monitored in the presence of an eNOS inhibitor and a cGMP inhibitor.Results:The percentage of adhesion was significantly lower in cirrhosis platelets. Time-dependent changes in the cGMP NO and NOS level in platelets stimulated with collagen were significantly high, with a significantly low level of elevation of cytosolic Ca2+in cirrhosis as adhesion proceeded. The results showed improved platelet adhesion with inhibitors of NOS and cGMP with concomitant elevation in Ca2+level.Conclusions:It is inferred that elevation in the formation of cGMP due to stimulation of NOS activity inhibits Ca2+mobilization from the internal store, an essential process to trigger platelet activation. The abnormal alterations were significantly lower in cirrhosis patients without bleeding complications. So, it could be stated that the bleeding abnormality in liver cirrhosis might be due to defective platelet adhesion influenced by the NO-cGMP pathway.

Combination of l-arginine supplementation and exercise performance on blood pressure responses in normotensive and hypertensive postmenopausal women: A crossover study

The purpose of this study was to evaluate the effects of l -arginine supplementation associated with exercise performance on the blood pressure (BP) regulation in normotensive (NT, n = 16) and hypertensive (HT, n = 7) postmenopausal women using a cross-over methodology. In four different days randomly, resting BP was measured and 9 g of l -arginine were given orally for the volunteers (Ajinomoto, Japan). Forty-five minutes after, the participants performed 30 min of exercise on treadmill at the maximal lactate steady state intensity. Blood venous was collected before the l -arginine supplementation and 45 min after the end of the exercise (ARG-EXE day). During the others three days, the volunteers performed exercise taking placebo pills (EXE day), or only l -arginine without exercise (ARG day), or did not take l -arginine nor performed exercise (CON day). Plasma Nitrite/Nitrate (NOx – Cayman, MI, USA) and cyclic guanosine monophosphate (cGMP – R&D systems, MN, USA) levels were measured by ELISA method. Women were overweight and non-diabetic. In NT women, systolic BP was 43% and 100% lower in EXE and ARG-EXE, respectively. Diastolic BP was 42% lower only in ARG-EXE comparing with CON day. No difference was found in NOx, but cGMP levels were higher in the ARG-EXE day (190%) comparing with CON day. in HT women, systolic BP was 70% and 40% lower in EXE and ARG-EXE days, respectively which was not accompanied by any changing in NOx or cGMP levels. In NT women, l -arginine plus exercise improve both systolic and diastolic BP that was positively associated with cGMP levels.

Altered expression of the natriuretic peptide system in genetically modified heme oxygenase-1 mice treated with high dietary salt

Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. With the natriuretic peptide system (NPS) as a key player in cardiovascular homeostasis and tissue dynamics, we sought to examine the effect of high dietary salt treatment in genetic models of HO-1 expression, and assessed the expression of the NPS in the left ventricle (LV), to determine if the effects of altered HO-1 expression may be due to modified levels of the NPS. Age-matched 12-week old male HO-1 knockout (HO-1(-/-)) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1(Tg)) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5weeks. LV mRNA expression was determined using quantitative real-time PCR. ANP peptide level was measured in the LV and plasma using radioimmunoassay, and LV cyclic 3'-5' guanosine monophosphate level was measured using an enzyme immunoassay kit. HO-1(-/-) fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1(+/+) mice fed NS diet. HO-1(-/-) mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no noticeable effect on LV/BW in HO-1(Tg) mice compared to controls. HO-1(Tg) mice had significantly higher ANP and BNP expression compared to controls. There were no differences in LV cGMP levels among all genotypes and dietary treatments. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. Both were substantiated by peptide levels as measured by RIA. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS.

Elevated plasma concentration of NO and cGMP may be responsible for the decreased platelet aggregation and platelet leukocyte conjugation in platelets hypo-responsive to catecholamines

Impaired responsiveness to epinephrine and other catecholamines (CA) were previously reported in platelets of 20 ∼ 30% healthy Japanese and Koreans. In the present study, the possible mechanisms of different responsiveness to CA in platelets of CA hypo-responders (CA-HY) and CA good-responders (CA-GR) were investigated. Increased platelet-leukocyte conjugate (PLC) formations were observed with whole blood of CA-GR than with that of CA-HY in both non-stimulated [mean fluorescence intensity (MFI) values: 1.33 ± 0.26 vs. 1.16 ± 0.19] and ADP (MFI: 5.54 ± 3.46 vs. 2.15 ± 1.13) or TRAP (MFI: 5.11 ± 2.32 vs. 3.38 ± 1.47) activated states. The platelets of CA-GR, when stimulated with ADP (10 µM), released approximately twice the amount of ATP than those of CA-HY (0.88 ± 0.65 and 0.45 ± 0.36 nmole, respectively). Nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were significantly higher in non-stimulated PRP of CA-HY than in that of CA-GR (70.3 ± 24.1 µM and 14.1 ± 4.9 nM vs. 41.1 ± 15.8 µM and 6.7 ± 2.4 nM, respectively). The platelet-monocyte conjugation induced with either ADP or TRAP was significantly reduced in CA-GR with the addition of linsidomine, a NO donor, (MFI: 2.78 ± 0.43 vs. 3.73 ± 0.90, or 4.28 ± 0.95 vs. 5.76 ± 1.33, respectively). Moreover, the degree of platelet aggregation and the ATP secretion induced by epinephrine in CA-GR were significantly retarded with the addition of either linsidomine or 8-Bromo-cGMP (a cGMP analog) with more substantial effects on ATP release than aggregation. The results suggested that elevated NO and/or cGMP plasma levels may be responsible for the lower platelet aggregation and PLC formation observed in CA-HY than that in CA-GR.

Thrombospondin 1 expression and angiogenesis in breast carcinoma and their relation with platelet activity

This study investigates angiogenesis and the expression of thrombospondin 1 in invasive ductal carcinoma of the breast and their possible relation to platelet counts and platelet activity. The study included 20 cases of invasive ductal carcinoma. Platelet activity was evaluated by determining thromboxane B2 and cyclic guanosine monophosphate (cGMP) levels by enzyme immunoassay (EIA).Thrombospondin (TSP) 1 and CD34 expression was studied immunohistochemically. Mean platelet count of the patient group was significantly greater than the mean platelet count of the control group (P < 0.05). The platelet counts were positively correlated with tumour size (r=0.609; P < 0.01). Platelet counts were higher in the patients who had grade 3 microvessel density (P < 0.05). The mean basal platelet cGMP level in the patient group was significantly lower than it was in the control group (P < 0.05). Focal TSP immunoreactivity was detectable in 5 (20%) cases in the tumour cells, and in 9 (45%) cases in the stroma. We did not find any correlation between TSP-1 staining and angiogenesis, platelet counts, platelet activity, and the histological prognostic parameters. Our study confirms the essential role of platelets in tumour growth and angiogenesis. Decreased levels of cGMP in the patient group may cause platelet hyperreactivity. Although thrombospondin 1 may be upregulated in malignant breast tissue, this is not sufficient for tumour growth and dissemination according to our results.

The Effect of Lipopolysaccharide from Proteus mirabilis on the Level of the Stable End Metabolic Products of Nitric Oxide in Blood Platelets

Nitric oxide ((*)NO) plays an important role in a number of physiologic processes. Evidence exists that (*)NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of (*)NO in platelets occurs. In these studies, production of the stable end-products of (*)NO-nitrite and nitrate (NO(x)) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO(x) in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO(x) level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO(x) level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate (*)NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).