NG-nitroarginine Methyl Ester Research Articles

Repeat administration of human umbilical cord mesenchymal stem cells improves left ventricular diastolic function in mice with heart failure with preserved ejection fraction

Currently, no therapy is proven to effectively improve heart failure with preserved ejection fraction (HFpEF). Although stem cell therapy has demonstrated promising results in treating ischemic heart disease, the effectiveness of treating HFpEF with human umbilical cord mesenchymal stem cells (hucMSCs) remains unclear. To answer this question, we administered hucMSCs intravenously (i.v.), either once or repetitively, in a mouse model of HFpEF induced by a high-fat diet and NG-nitroarginine methyl ester hydrochloride. hucMSC treatment improved left ventricular diastolic dysfunction, reduced heart weight and pulmonary edema, and attenuated cardiac modeling (inflammation, interstitial fibrosis, and hypertrophy) in HFpEF mice. Repeat hucMSC administration had better outcomes than a single injection. In vitro, hucMSC culture supernatants reduced maladaptive remodeling in neonatal-rat cardiomyocytes. Ribonucleic acid sequencing and protein level analysis of left ventricle (LV) tissues suggested that hucMSCs activated the protein kinase B (Akt)/forkhead box protein O1 (FoxO1) signaling pathway to treat HFpEF. Inhibition of this pathway reversed the efficacy of hucMSC treatment. In conclusion, these findings indicated that hucMSCs could be a viable therapeutic option for HFpEF.

Combined vaccines against angiotensin II receptor type 1 and alpha 1D-adrenergic receptor for hypertension.

Compared with monotherapy, combination therapy with multiple antihypertensive drugs has demonstrated superior efficacy in the management of hypertension. The aim of this study was to explore the efficacy of multitarget combined vaccines in achieving simultaneous antihypertensive and target organ protection effects. Our team has developed ATRQβ-001 and ADRQβ-004 vaccines targeting Ang II type 1 receptor (AT1R) and α1D-adrenergic receptor (α1D-AR), respectively. In NG-nitroarginine methyl ester ( l -NAME) + abilities spontaneously hypertensive rats (SHRs) model, SHRs were simultaneously inoculated with ATRQβ-001 and ADRQβ-004 vaccines. Histological and biochemical analyses were performed to evaluate the antihypertensive effects and target organ protection of the ATRQβ-001 and ADRQβ-004 combined vaccines in comparison with those of the single vaccine. Both ATRQβ-001 and ADRQβ-004 vaccines induced robust antibody production, resulting in persistent high antibody titers in rats. Notably, the combined administration of both vaccines significantly decreased SBP in SHRs compared with treatment with a single vaccine, both before and after l -NAME administration. Furthermore, the combined vaccine regimen demonstrated superior efficacy in protecting against vascular remodeling, myocardial hypertrophy and fibrosis, and kidney injury in SHRs. Mechanistically, the combined vaccines exhibited significantly downregulated the expression of angiotensin II type 1 receptor (AT1R) and α1D-adrenergic receptor (α1D-AR). Importantly, no apparent immune-related adverse effects were observed in animals immunized with the combined vaccines. Preliminary findings from this investigation suggest that co-administration of the novel ATRQβ-001 and ADRQβ-004 vaccines holds potential as a groundbreaking therapeutic strategy for managing hypertension.

Gut microbiota-derived trimethylamine N-Oxide: a novel target for the treatment of preeclampsia

ABSTRACT Pre-eclampsia (PE) is the most common complication of pregnancy and seriously threatens the health and safety of the mother and child. Studies have shown that an imbalance in gut microbiota can affect the progression of PE. Trimethylamine N-oxide (TMAO) is an intestinal microbiota-derived metabolite that is thought to be involved in the occurrence of PE; however, its causal relationship and mechanism remain unclear. In this clinical cohort study, including 28 patients with eclampsia and 39 matched healthy controls, fecal samples were collected for 16S rRNA gene sequencing, and serum was collected for targeted metabolomics research. The results showed that the level of TMAO and the abundance of its source bacteria had significantly increased in patients with PE, and were positively correlated with the clinical progression of PE. Fecal microbiota transplantation (FMT) was applied to an antibiotic-depleted-treated mouse model and targeted inhibition of TMAO. The results of the FMT experiment revealed that mice that received fecal microbiota transplantation from patients with PE developed typical PE symptoms and increased oxidative stress and inflammatory damage, both of which were reversed by 3,3-Dimethyl-1-butanol (DMB), a TMAO inhibitor, which also improved pregnancy outcomes in the model mice. Similar results were obtained in the classical NG-Nitroarginine methyl ester (L-NAME) induced PE mouse model. Mechanistically, TMAO promotes the progression of PE by regulating inflammatory and oxidative stress-related signaling pathways, affecting the migration and angiogenesis of vascular endothelial cells, as well as the migration and invasion of trophoblast cells. Our results reveal the role and mechanism of gut microbiota and TMAO in the progression of PE, provides new ideas for exploring the pathogenesis and therapeutic targets of PE, and determines the potential application value of TMAO as a target for PE intervention.

Reduced Brain Cortex Angiogenesis in the Offspring of the Preeclampsia-Like Syndrome.

Children from pregnancies affected by preeclampsia have an increased risk of cognitive and behavioral alterations via unknown pathophysiology. We tested the hypothesis that preeclampsia generated reduced brain cortex angiogenesis in the offspring. The preeclampsia-like syndrome (PELS) mouse model was generated by administering the nitric oxide inhibitor NG-nitroarginine methyl ester hydrochloride. Confirmatory experiments were done using 2 additional PELS models. While in vitro analysis used mice and human brain endothelial cells exposed to serum of postnatal day 5 pups or umbilical plasma from preeclamptic pregnancies, respectively. We report significant reduction in the area occupied by blood vessels in the motor and somatosensory brain cortex of offspring (postnatal day 5) from PELS compared with uncomplicated control offspring. These data were confirmed using 2 additional PELS models. Furthermore, circulating levels of critical proangiogenic factors, VEGF (vascular endothelial growth factor), and PlGF (placental growth factor) were lower in postnatal day 5 PELS. Also we found lower VEGF receptor 2 (KDR [kinase insert domain-containing receptor]) levels in mice and human endothelial cells exposed to the serum of postnatal day 5 PELS or fetal plasma of preeclamptic pregnancies, respectively. These changes were associated with lower in vitro angiogenic capacity, diminished cell migration, larger F-actin filaments, lower number of filopodia, and lower protein levels of F-actin polymerization regulators in brain endothelial cells exposed to serum or fetal plasma of offspring from preeclampsia. Offspring from preeclampsia exhibited diminished brain cortex angiogenesis, associated with lower circulating VEGF/PlGF/KDR protein levels, impaired brain endothelial migration, and dysfunctional assembly of F-actin filaments. These alterations may predispose to structural and functional alterations in long-term brain development.

Is Glyceryl Trinitrate, a Nitric Oxide Donor Responsible for Ameliorating the Chemical-Induced Tissue Injury In Vivo?

Oxidative stress induced by well-known toxins including ferric nitrilotriacetate (Fe-NTA), carbon tetrachloride (CCl4) and thioacetamide (TAA) has been attributed to causing tissue injury in the liver and kidney. In this study, the effect of glyceryl trinitrate (GTN), a donor of nitric oxide and NG-nitroarginine methyl ester (l-NAME), a nitric oxide inhibitor on TAA-induced hepatic oxidative stress, GSH and GSH-dependent enzymes, serum transaminases and tumor promotion markers such as ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats were examined. The animals were divided into seven groups consisting of six healthy rats per group. The six rats were injected intraperitoneally with TAA to evaluate its toxic effect, improvement in its toxic effect if any, or worsening in its toxic effect if any, when given in combination with GTN or l-NAME. The single necrogenic dose of TAA administration caused a significant change in the levels of both hepatic and serum enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), γ-glutamyl transpeptidase (GGT), glucose 6-phosphate dehydrogenase (G6PD), alanine aminotransferase (AST) and aspartate aminotransferase (ALT). In addition, treatment with TAA also augmented malondialdehyde (MDA), ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats liver. Concomitantly, TAA treatment depleted the levels of GSH. However, most of these changes were alleviated by the treatment of animals with GTN dose-dependently. The protective effect of GTN against TAA was also confirmed histopathologically. The present data confirmed our earlier findings with other oxidants including Fe-NTA and CCl4. The GTN showed no change whatsoever when administered alone, however when it was given along with TAA then it showed protection thereby contributing towards defending the role against oxidants-induced organ toxicity. Overall, GTN may contribute to protection against TAA-induced oxidative stress, toxicity, and proliferative response in the liver, according to our findings.

Therapeutic role of nitroglycerin against copper-nitrilotriacetate induced hepatic and renal damage.

Earlier we have shown that exposure to copper-nitrilotriacetate (Cu-NTA) manifests toxicity by generating oxidative stress and potent induction of proliferative reaction in the liver and kidney. In the study, we look at the impact of nitroglycerin (GTN) administration on Cu-NTA-induced oxidative stress and hyperproliferative response in the liver and kidney. GTN administration intraperitoneally to male Wistar rats after Cu-NTA administration intraperitoneally caused substantial protection against Cu-NTA-induced tissue injury, oxidative stress and hyperproliferative response. Cu-NTA administration at a dose of 4.5mg/kg body weight produces significant (p < .001) elevation in biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CREA) with a concomitant increase in microsomal lipid peroxidation. Along with these alterations, we discovered a substantial increment in [3H]thymidine incorporation into hepatic and renal DNA synthesis (p < .001). Cu-NTA-induced tissue damage and lipid peroxidation in hepatic and renal tissues were inhibited by GTN treatment in a dose-dependent manner (p < .05-0.001). Furthermore, GTN can suppress the hyperproliferative response elicited by Cu-NTA by down-regulating the rate of [3H]thymidine incorporation into hepatic and renal DNA (p < .01-0.001). Protective effect of GTN against Cu-NTA was also confirmed by histopathological changes in liver and kidney. This result suggests that GTN may serve as a scavenger for reactive oxygen species (ROS) and reduces toxic metabolites of Cu-NTA, thereby avoiding tissue injury and oxidative stress. Further, administration of NO inhibitor, NG-Nitroarginine methyl ester (L-NAME), exacerbated Cu-NTA induced oxidative tissue damage and cell proliferation. Overall, GTN reduces Cu-NTA-induced tissue damage, oxidative stress, and proliferative response in the rat liver and kidney, according to these findings. On the basis of the above results, present study suggests that GTN may be a potential therapeutic agent for restoration of oxidative damage and proliferation to liver and kidney.

Anti-nociceptive Activity of Quebracho tannin Extract on Pain Induced by Formalin and Writhing Tests in Mice

Background: Based on positive role of the tannins for pain relief, there is no report for possible antinociceptive activity of the Quebracho tannin. Objectives: This study aimed to determine the anti-nociceptive activity of the Quebracho tannin extract (QTE) on pain in mice. Materials and Methods: For this purpose, 340 mice were used for formalin and writhing tests each including 4 experiments with 4 sub-groups. In experiment 1, mice were injected with saline, QTE (100 mg/kg), QTE (200 mg/kg), QTE (400 mg/kg), and morphine (5 mg/kg). In the second experiment, injections included saline, QTE (400 mg/kg), naloxone (2 mg/kg), and QTE + naloxone. Experiments 3 and 4 were similar to experiment 2, except that mice injected were with NG-nitro arginine methyl ester (L-NAME, 10 mg/kg) and cyproheptadine (4 mg/kg) instead of naloxone. Then, formalin (1%) was injected, and time spent for licking the injected paw was recorded until 30 minutes following injection in the first and second phases. Finally, injections in 4 experiment groups were the same, and animals were intraperitoneally injected with acetic acid, and contractions were recorded in the writhing test category. Results: According to the results, QTE (100, 200, and 400 mg/kg) decreased pain in the injected paw (P=0.001) and inhibited the pain response by 59.37% (P=0.001). Moreover, the injection of naloxone + QTE significantly decreased pain in the injected paw (P=0.021). Eventually, the injection of the L-NAME + QTE significantly reduced the anti-nociception effect of the QTE on the formalin test (P=0.031) and writhing contractions (55.75%, P=0.033). Conclusion: These findings suggested anti-nociceptive properties of the QTE mediated by opioidergic and nitrergic systems.

Protective effects of anandamide against cisplatin-induced peripheral neuropathy in rats

Cisplatin (CIS) is an effective antineoplastic agent used in the treatment of several cancer types. Peripheral neuropathy is a major dose-limiting side-effect in CIS therapy. Cannabinoids may alleviate this painful side effect. This study investigated the analgesic effects of anandamide (AN) on CIS-induced peripheral neuropathy, in vitro effects of AN in CIS neurotoxicity, and the contribution of nitric oxide (NO) in this effect. This is an experimental animal study. Primary dorsal root ganglion (DRG) cultures were prepared from one-day-old rats for in vitro investigations. DRG cells were incubated with CIS (100–300 M), and AN (10, 50, 100, and 500 μM) was administered with the submaximal concentration of CIS. Female Sprague Dawley rats were divided into control, CIS, CIS+AN, CIS+AN+L-NG-nitro arginine methyl ester (LNAME). CIS was administered 3 mg/kg i.p once weekly for 5 weeks. AN (1 mg/kg i.p) or in combination with 10 mg/kg i.p LNAME was administrated 30 min before CIS injection. Mechanical allodynia, thermal hyperalgesia, and tail clip tests were performed. After intracardiac perfusion, sciatic nerves (SN), and DRGs were isolated and semi-thin sections were stained with toluidine blue and investigated histologically. SPSS v. 21.0 and Sigma STAT 3.5 were used for statistical analysis. One/two way ANOVA, Kruskal–Wallis, and Wilcoxon signed ranks tests were used. A p-value of 0.05 was accepted as significant. CIS caused significant mechanical allodynia. AN and AN+LNAME significantly increased hind paw withdrawal latency in mechanical allodynia test. The degenerated axons significantly increased in CIS group, while decreased in AN group. The frequency of larger neurons seemed to be higher in CIS+AN group. AN may be a therapeutic alternative for the treatment of CIS-induced peripheral neuropathy. However, its central adverse effects must be considered.

Gentiopicroside Produces Endothelium-Independent Vasodilation by Deactivating the PI3K/Akt/Rho-Kinase Pathway in Isolated Rat Thoracic Aorta.

Gentiopicroside (GPS), a main active secoiridoid glucoside derived from the roots of perennial herbs in the Gentianaceae family, has antispasmodic and relaxant effects. However, the vasorelaxant effects of GPS on aortic rings and the molecular mechanisms involved in these effects are not yet clear. Therefore, we investigated whether GPS inhibits phenylephrine- (PE-) or KCl-induced contractions in isolated rat thoracic aortic rings. The present study found that GPS produced a dose-dependent relaxation in aortic rings precontracted with PE or KCl and significantly reduced CaCl2-, narciclasine- (Rho-kinase activator-), and phorbol-12,13-diacetate- (PKC activator-) induced vasocontractions. Pretreatment with NG-Nitroarginine methyl ester hydrochloride (L-NAME, NOS inhibitor), methylene blue (sGC inhibitor), indomethacin (COX inhibitor), 4-aminopyridine (KV channel inhibitor), and glibenclamide (KATP channel inhibitor) had no influence on the vasorelaxant effect of GPS, while BaCl2 (Kir channel inhibitor), tetraethylammonium chloride (KCa channel inhibitor), ruthenium red (RYR inhibitor), and heparin (IP3R inhibitor) significantly reduced GPS-induced vasorelaxation. Moreover, GPS pretreatment remarkably inhibited the influx of Ca2+ in vascular smooth muscle cells stimulated using KCl or PE-containing CaCl2 solution. Western blot analysis confirmed that GPS treatment inhibited PE-induced increases in the protein levels of p-Akt, p-myosin light chain (MLC), and p-myosin-binding subunit of myosin phosphatase 1 (MYPT1) in the aortic rings. Additionally, the vasorelaxation activity of GPS was attenuated upon pretreatment with LY294002 (PI3K/Akt inhibitor), Y27632 (Rho-kinase inhibitor), and verapamil (L-type Ca2+ channel inhibitor). These findings demonstrate that GPS exhibits endothelium-independent vasorelaxant effects through inhibition of voltage-dependent, receptor-operated, and inositol triphosphate receptor (IP3R)/ryanodine receptor- (RYR-) mediated Ca2+ channels as well as the PI3K/Akt/Rho-kinase signaling pathway.

Locomotor stimulation by acute propofol administration in rats: Role of the nitrergic system.

The addictive potential of propofol has been scientifically discussed. Drugs' psychostimulant properties that can be assessed via measurements of locomotor activity are linked to their addictive properties. No studies that have investigated the effects of propofol on locomotor activity have been reported to date. The present study sought to investigate the effects and possible mechanisms of action of propofol on locomotor activity in rats. Adult male albino Wistar rats (250-330g) were used as subjects. The locomotor activities of the rats were recorded for 30min immediately following intraperitoneal administration of propofol (20 and 40mg/kg), saline or vehicle (n=8 for each group). NG-nitro arginine methyl ester (l-NAME, 15-60mg/kg), a nitric oxide (NO) synthase inhibitor, and haloperidol (0.125-5mg/kg), a non-specific dopamine receptor antagonist, were also administered to other groups of rats 30min prior to the propofol (40mg/kg) injections, and locomotor activity was recorded for 30min immediately after propofol administration (n=8 for each group). Propofol produced significant increases in the locomotor activities of the rats in the first 5min of the observation period [F(2,21)=9.052; p<0.001]. l-NAME [F(4,35)=3.112; p=0.02] but not haloperidol [F(4,35)=2.440; p=0.067] pretreatment blocked the propofol-induced locomotor hyperactivity. l-NAME did not cause any significant change in locomotor activity in naïve rats [F(2,21)=0.569; p=0.57]. Our results suggest that propofol might cause a short-term induction of locomotor activity in rats and that this effect might be related to nitrergic but not dopaminergic mechanisms.

Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

PurposeThe location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits.Materials and MethodsIsolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester, 7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated.ResultsIn the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10-9 to 10-4 M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 µM L-NAME or 10 µM 7-nitroindazole) but also by 10 µM atropine and some selective muscarinic receptor antagonists (10-6 M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10-6 M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine.ConclusionsAcetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

The effects and molecular mechanisms of simvastatin on liver fibrosis in rats with non-alcoholic fatty liver disease

Objective To investigate the effects and molecular mechanism of simvastatin in liver fibrosis model of non-alcoholic fatty liver disease (NAFLD) in vivo and hepatic stellate cell in vitro.Methods Firstly,the rat liver fibrosis model of NAFLD was established by high-fat diet administration and intervened with simvastatin.The expression of endothelial nitric oxide synthase (eNOS),inducible nitric oxide synthase (iNOS) and Collagen Ⅰ at mRNA and protein level were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Secondly,quiescent phenotype of LX-2 cell line was induced by promoting adipocyte differentiation medium in vitro,and then the quiescent phenotype of LX-2 cell were treated with transforming growth factor β1(TGF-β1),Nitroso-L-arginine methyl ester (L-NAME) which was NOS inhibitor,simvastatin,TGFβ1 with simvastatin,and L-NAME with simvastatin separately.The changes of eNOS,iNOS,αsmooth muscle actin (α-SMA) and Collagen Ⅰ expressions at mRNA and protein level were determined by RT-PCR and Western blot.Results As modeling time extended,the expressions of eNOS in rat's liver tissue of model group at mRNA and protein level decreased gradually,however the expression of iNOS and Collagen Ⅰ at mRNA and protein level increased gradually,compared with normal control group and the differences were statistically significant (P ＜0.05 and 0.01).By 24weeks,the expressions of eNOS in rat's liver tissue of simvastatin group at mRNA and protein level were increased,the expression of iNOS at mRNA and protein level were decreased and the expression of Collagen Ⅰ at mRNA and protein level were decreased (all P ＜0.05).The expression of eNOS in rat's liver tissue of model group negatively correlated with the expression of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).The expression of iNOS positively correlated with that of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).In LX-2 cell culture,L-NAME inhibited the activation of LX-2,reduced eNOS and iNOS expression and increased α-SMA and Collagen Ⅰ expression,consistent with the role of TGF-β1.Simvastatin could directly increase the eNOS expression both in quiescent and activated LX-2 cells,decrease iNOS expression,maintain quiescent phenotype and inhibit its activation.Conclusions Simvastatin ameliorated the genesis and progression of liver fibrosis by increasing eNOS expression in LX-2 cells and reducing iNOS,α-SMA and Collagen Ⅰ expression. Key words: Simvastatin; Liver cirrhosis; Nitric oxide synthase; Astrocytes; NG-nitroarginine methylester; Disease models, animal

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10–20 dyn/cm2) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm2 had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-NG-monomethyl arginine (l-NMMA) and l-NG-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.

Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

Blood pressure and renal blow flow responses in heme oxygenase-2 knockout mice

Heme oxygenase (HO) is the enzyme responsible for the breakdown of heme-generating carbon monoxide (CO) and biliverdin in this process. HO-2 is the constitutively expressed isoform in most tissues, such as the kidney and vasculature. CO generated by HO is believed to be an important vasodilator in the renal circulation along with another gas, nitric oxide (NO). To determine the importance of HO-2 in the regulation of blood pressure and renal blood flow (RBF), we treated HO-2 knockout (KO) mice chronically with either ANG II or N(G)-nitroarginine methyl ester (l-NAME). Basal blood pressures were not different between wild-type (WT), heterozygous (HET), or KO mice and averaged 113 +/- 3 vs. 115 +/- 2 vs. 116 +/- 2 mmHg. Similar increases in blood pressure to chronic ANG II as well as l-NAME treatment were observed in all groups with blood pressures increasing an average of 30 mmHg in response to ANG II and 15 mmHg in response to l-NAME. Basal RBFs were not different between the groups averaging 6.0 +/- 0.5 (n = 6) vs. 4.8 +/- 0.6 (n = 10) vs. 5.8 +/- 0.7 (n = 6) ml*min(-1)*g(-1) kidney weight in WT, HET, and KO mice. HO-2 KO and HET mice exhibited an attenuated decrease in RBF in response to acute administration of ANG II, while no differences were observed with l-NAME. Our data indicate that blood pressure and RBF responses to increased ANG II or inhibition of nitric oxide are not significantly enhanced in HO-2 KO mice.

Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.

Role of Scavenger Receptor Class B Type I and Sphingosine 1-Phosphate Receptors in High Density Lipoprotein-induced Inhibition of Adhesion Molecule Expression in Endothelial Cells

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S1P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P(1), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P(3) receptors severely suppressed the stimulatory actions of S1P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P(3) receptors to G(12/13) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P(3) receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S1P receptors, especially S1P(1).

Effect of Acidosis on Isolated Porcine Retinal Vessels

Purpose: To study the effect of normocapnic (NA) and hypercapnic acidosis (HA) on the tone, the intracellular calcium level ([Ca2 +]i), and the membrane potential of smooth muscle cells in porcine retinal arterioles. Methods: Twenty-four porcine retinal arterioles were mounted in a wire myograph for isometric recording of the wall tension. The vessels were precontracted with 0.3 μ M U46619 and were exposed to NA (pH = 7.0) and HA (pH = 7.0). Intracellular calcium was measured using the fluorophore Fura-2AM (n = 12). In six vessels, 0.1 mM NG-nitroarginine methyl ester (L-NAME) was added to block NO synthesis. The membrane potential of smooth muscles cells was measured in situ with sharp glass electrodes (n = 12). Results: NA and HA induced both a decrease in wall tension from 1.04 ± 0.06 N/m to 0.65 ± 0.1 N/m (p < 0.01) (NA) and 0.56 ± 0.1 N/m (p < 0.01) (HA) and a decrease in [Ca2 +]i as evidenced from the change in the Fura-2 fluorescence emission ratio from 0.66 ± 0.03 to 0.57 ± 0.05 (p = 0.005) (NA) and 0.56 ± 0.05 (p = 0.002) (HA). These results were unaffected by inhibition of NO-synthesis. NA and HA also both induced hyperpolarization of the smooth muscle membrane from −18 ± 0.7 mV during precontraction to −26 ± 1.9 mV (p = 0.002) (NA) and −24 ± 2.6 mV (p = 0.02) (HA). Conclusions: Acidosis-induced relaxation of the tone in preconstricted isolated porcine retinal arterioles is associated with a decrease in intracellular calcium and a hyperpolarization of the smooth muscle cells. The acidosis-induced relaxation is independent of CO2 and is not mediated through NO.

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases.

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.

Role of nitric oxide and β-adrenoceptors of the central nervous system on the salivary flow induced by pilocarpine injection into the lateral ventricle

Our studies have focused on the effect of l- NG-nitroarginine methyl ester ( l-NAME), an inhibitor of nitric oxide synthase (NOS), and l-arginine, the substrate of NOS, on salivary secretion induced by the administration of pilocarpine into the lateral cerebral ventricle (LV) of rats. The present study has also investigated the role of the β-adrenergic agonists and antagonist injected into LV on the salivary secretion elicited by the injection of pilocarpine into LV. Male Holtzman rats with a stainless-steel cannula implanted into the LV were used. The amount of salivary secretion was studied over a 7-min period after injection of pilocarpine, isoproterenol, propranolol, salbutamol, salmeterol, l-NAME and l-arginine. The injection of pilocarpine (10, 20, 40, 80 and 160 μg/μl) into LV produced a dose-dependent increase in salivary secretion. The injection of l-NAME (40 μg/μl) into LV alone produced an increase in salivary secretion. The injection of l-NAME into LV previous to the injection of pilocarpine produced an increase in salivary secretion. l-Arginine (30 μg/μl) injected alone into LV produced no change in salivary secretion. l-Arginine injected into LV attenuated pilocarpine-induced salivary secretion. The isoproterenol (40 nmol/μl) injected into LV increased the salivary secretion. When injected previous to pilocarpine at a dose of 20 and 40 μg/μl, isoproterenol produced an additive effect on pilocarpine-induced salivary secretion. The 40-nmol/μl dose of propranolol injected alone or previous to pilocarpine into LV attenuated the pilocarpine-induced salivary secretion. The injection of salbutamol (40 nmol/μl), a specific β-2 agonist, injected alone into LV produced no change in salivary secretion and when injected previous to pilocarpine produced an increase in salivary secretion. The 40-nmol/μl dose of salmeterol, a long-acting β-2 agonist, injected into LV alone or previous to pilocarpine produced no change in salivary secretion. The results have shown that central injections of l-NAME and l-arginine interfere with the salivary secretion, which implies that might participate in pilocarpine-induced salivary secretion. The interaction between cholinergic and β-adrenergic receptors of the central nervous system (CNS) for the control of salivary secretion can also be postulated.