Oleanolic acid induces relaxation and calcium-independent release of endothelium-derived nitric oxide

Background and purpose:

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO).

Experimental approach:

Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs).

Key results:

Oleanolic acid (3–30 μM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 μM) and by the NO scavenger, oxyhaemoglobin (10 μM). Oleanolic acid-evoked NO increases were not reduced in Ca²⁺-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 μM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine⁴⁷³ (Akt-Ser⁴⁷³) and eNOS at Serine¹¹⁷⁷ (eNOS-Ser¹¹⁷⁷), which was attenuated by inhibitors of phosphoinositide-3-kinase.

Conclusions and implications:

This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser⁴⁷³ followed by phosphorylation of eNOS-Ser¹¹⁷⁷.     

Effect of oral organic nitrates on expression and activity of vascular soluble guanylyl cyclase

Background and purpose:

The regulation of vascular soluble guanylyl cyclase (sGC) expression by nitric oxide (NO) is still under discussion. In vitro, NO has been shown to downregulate the expression of sGC but it is unclear if this mechanism is operative in vivo and occurs during nitrate treatment.

Experimental approach:

We investigated whether high dose isosorbide mononitrate (ISMN) or pentaerythrityl tetranitrate (PETN) treatment changes vascular sGC expression and activity in vivo. New Zealand White rabbits received a standard diet, 2 or 200 mg ISMN kg⁻¹ d⁻¹ for 16 weeks, and C57BL/6 mice received a standard diet, 6, 60 or 300 mg PETN kg⁻¹ d⁻¹ for four weeks. Absorption was checked by measuring the plasma levels of the drug/metabolite.

Key results:

Western blots of rabbit aortic rings showed similar protein levels of sGC α1- (P=0.2790) and β1-subunits (P=0.6900) in all groups. Likewise, ANOVA showed that there was no difference in the expression of sGC in lungs of PETN-treated mice (P=0.0961 for α1 and P=0.3709 for β1). The activities of isolated sGC in response to SNAP (1 μM–1 mM) were identical in aortae of ISMN-treated rabbits (P=0.0775) and lungs of PETN-treated mice (P=0.6348). The aortic relaxation response to SNAP slightly decreased at high ISMN but not at high PETN.

Conclusions and implications:

These data refute the hypothesis that therapeutic treatment with long acting NO donors has a significant impact on the regulation of vascular sGC expression and activity in vivo.     

Polymorphisms of genes in nitric oxide-forming pathway associated with ischemic stroke in Chinese Han population

Aim:

To investigate the association of polymorphisms in four critical genes implicated in the NO-forming pathway with ischemic stroke (IS) in a Chinese Han population.

Methods:

DNA samples of 558 IS patients and 557 healthy controls from Chinese Han population were genotyped using the Taqman^TM 7900HT Sequence Detection System. Six SNPs (rs841, rs1049255, rs2297518, rs1799983, rs2020744, rs4673) of the 4 related genes (eNOS, iNOS, GCH1, and CYBA) in the NO forming pathway were analyzed using the SPSS 13.0 software package for Windows.

Results:

One SNP located in the intron of GCH1 (rs841) was associated with IS independent of the traditional cardiovascular risk factors in co-dominant and dominant models (P=0.003, q=0.027; P=0.00006, q=0.0108; respectively). Moreover, the combination of rs1049255 CC+CT and rs841 GA+AA genotypes was associated with significantly higher risk for IS after adjustments (OR=1.73, 95% CI: 1.27–2.35, P<0.0001, q<0.0001).

Conclusion:

The data suggest that genetic variants within the NO-forming pathway alter susceptibility to IS in Chinese Han population. Replication of the present results in other independent cohorts is warranted.     

Chronic administration of BMS309403 improves endothelial function in apolipoprotein E-deficient mice and in cultured human endothelial cells

BACKGROUND AND PURPOSE

Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE^−/−) mice and in cultured human endothelial cells.

EXPERIMENTAL APPROACH

A-FABP was measured in aortae of ApoE^−/−mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae

KEY RESULTS

A-FABP was expressed in aortic endothelium of ApoE^−/− mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α₂-adrenoceptor agonist) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE^−/− mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403.

CONCLUSIONS AND IMPLICATIONS

Elevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.     

Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism

AIMS

Oral L-arginine supplementation has been used in several studies to improve endothelium-dependent, nitric oxide (NO)-mediated vasodilation. L-Arginine treatment is hampered by extensive presystemic elimination due to intestinal arginase activity. In contrast, L-citrulline is readily absorbed and at least in part converted to L-arginine. The aim of our study was to assess this metabolic conversion and its subsequent pharmacodynamic effects.

METHODS

In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers received six different dosing regimes of placebo, citrulline, and arginine. Pharmacokinetic parameters (C_max, T_max, C_min, AUC) were calculated after 1 week of oral supplementation. The ratio of plasma L-arginine over asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase (arginine/ADMA ratio), urinary cyclic guanosine monophosphate (cGMP) and nitrate excretion rates, and flow-mediated vasodilation (FMD) was measured to assess pharmacodynamic effects.

RESULTS

L-Citrulline dose-dependently increased AUC and C_max of plasma L-arginine concentration more effectively than L-arginine (P < 0.01). The highest dose of citrulline (3 g bid) increased the C_min of plasma L-arginine and improved the L-arginine/ADMA ratio from 186 ± 8 (baseline) to 278 ± 14 [P < 0.01, 95% confidence interval (CI) 66, 121]. Moreover, urinary nitrate and cGMP were increased from 92 ± 10 to 125 ± 15 µmol mmol⁻¹ creatinine (P = 0.01, 95% CI 8, 58) and from 38 ± 3.3 to 50 ± 6.7 nmol mmol⁻¹ creatinine (P = 0.04, 95% CI 0.4, 24), respectively. No treatment improved FMD over baseline. However, pooled analysis of all FMD data revealed a correlation between the increase of arginine/ADMA ratio and improvement of FMD.

CONCLUSION

Our data show for the first time that oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• L-Arginine is a semiessential amino acid that is converted to nitric oxide (NO) by NO synthase (NOS).
• NO improves endothelial function by elevating cyclic guanosine monophosphate.
• However, oral L-arginine treatment in humans is hampered by extensive metabolism.

WHAT THIS STUDY ADDS

• Oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner.
• L-Citrulline may thus be an alternative to L-arginine in patients with impaired NOS activity.

     

A ketolide antibiotic, telithromycin, inhibits vascular adrenergic neurotransmission in the rat mesenteric vascular bed

Background and purpose:

A ketolide antibiotic, telithromycin, has side effects including temporary loss of consciousness in clinical use, but the underlying mechanisms remain unclear. This study investigated the effects of telithromycin on perivascular nerve function in rat mesenteric arteries, in comparison with those of macrolide (erythromycin and clarithromycin) and new quinolone antibiotics (levofloxacin and gatifloxacin).

Experimental approach:

In vitro, vascular responses and release of noradrenaline induced by periarterial nerve stimulation (PNS) of rat perfused mesenteric vascular beds were measured in the presence of each antibiotic. In vivo blood pressure measurement was performed in Wistar rats.

Key results:

In mesenteric preparations with resting tone, telithromycin (10 nM–10 μM) markedly inhibited PNS (4–12 Hz)-induced adrenergic nerve- and exogenous noradrenaline-mediated vasoconstriction, whereas the other antibiotics slightly inhibited PNS-induced responses without affecting noradrenaline-induced responses. Telithromycin significantly reduced PNS (12 Hz)-evoked noradrenaline release in the perfusate. In pre-constricted preparations with or without endothelium, telithromycin (0.1 nM–10 μM) caused a concentration-dependent vasodilation. Telithromycin (10 nM) inhibited calcium-induced vasoconstriction in high KCl and calcium-free medium. None of the antibiotics used affected PNS (0.5–2 Hz)-induced calcitonin gene-related peptide (CGRP) nerve- and exogenous CGRP-mediated vasodilation. Intravenous injection of telithromycin significantly lowered blood pressure in anaesthetized rats.

Conclusions and implications:

These results suggest that telithromycin causes not only strong inhibition of perivascular adrenergic neurotransmission but also a vasodilator action in mesenteric vascular beds and hypotension. It is thus possible that telithromycin increases visceral blood flow, consequently reducing cerebral blood flow and resulting in a temporary loss of consciousness.     

Arginase II inhibition prevents nitrate tolerance

BACKGROUND AND PURPOSE

Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance.

EXPERIMENTAL APPROACH

Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII –/–) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence.

KEY RESULTS

Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration–response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII –/– mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII –/– mice did not.

CONCLUSION AND IMPLICATIONS

Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.     

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium-dependent NO signalling

BACKGROUND AND PURPOSE

Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

EXPERIMENTAL APPROACH

Cumulative relaxation responses to obestatin peptides were assessed in rat isolated aorta and mesenteric artery (n≥ 8) in the presence and absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC).

KEY RESULTS

Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1–23) were greater than those to obestatin(1–10) and obestatin(11–23). Obestatin(1–23)-induced relaxation was attenuated by endothelial denudation, l-NAME (NOS inhibitor), high extracellular K⁺, GDP-β-S (G-protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK_Ca blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked GPCR, PI3K/PKB, Ca²⁺-dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K⁺. Supporting data from BAEC indicated that nitrite production, intracellular Ca²⁺ and PKB phosphorylation were increased after exposure to obestatin(1–23). Relaxations to obestatin(1–23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarizing factors (EDHFs) and combined SK_Ca/IK_Ca blockade, suggesting that EDHF-mediated pathways were not involved.

CONCLUSIONS AND IMPLICATIONS

Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterized by endothelial dysfunction and cardiovascular complications.     

Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta

Background and purpose:

Previous work has shown that N^G-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N^G, N^G-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.

Experimental approach:

The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.

Key results:

ADMA (100 µM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300–1000 µM) had no greater effect. Although ADMA (100 µM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 µM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 µM), protected ACh-induced relaxation against blockade by l-NAME (30 µM).

Conclusions and implications:

In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA.     

Acute activation of eNOS by statins involves scavenger receptor-B1, G protein subunit Gi, phospholipase C and calcium influx

Background and purpose:

Statins (HMG CoA reductase inhibitors) have beneficial effects independent of reducing cholesterol synthesis and this includes their ability to acutely activate endothelial nitric oxide synthase (eNOS). The mechanism by which this occurs is largely unknown and thus we characterized the pathways by which statins activate NOS, including involvement of scavenger receptor-B1 (SR-B1), which is expressed in endothelial cells and maintains cholesterol concentrations.

Experimental approach:

Nitric oxide production was monitored in bovine aortic endothelial cells (BAECs) exposed to lovastatin (LOV) or pravastatin (PRA) for 10–20 min, alone or following pre-exposure to the end product of HMG-CoA reductase (mevalonate), G protein inhibitors (pertussis/cholera toxins), phospholipase C (PLC) inhibitor (U-73122), or intracellular and extracellular calcium chelators – BAPTA-AM and EGTA (respectively), or a function blocking antibody to SR-B1.

Key results:

Both statins increased NO production in a rapid, dose-dependent and HMG-CoA reductase-independent manner. Inhibiting Gi protein or PLC almost completely blocked statin-induced NO generation. Additionally, removing extracellular calcium inhibited statin-induced NO production. COS-7 cells co-transfected with eNOS and SR-B1 increased NO production when exposed to LOV or high-density lipoprotein (HDL), an agonist of SR-B1. These effects were not observed in COS-7 cells with eNOS alone or co-transfected with bradykinin receptor 2, indicating specificity for SR-B1. Further, pretreatment of BAEC with blocking antibody for SR-B1 blocked NO responses to statins and HDL.

Conclusions and implications:

LOV and PRA acutely activate eNOS through pathways that include the cell surface receptor SR-B1, Gi protein, phosholipase C and entry of extracellular calcium into endothelial cells.     

Reversal of SIN-1-induced eNOS dysfunction by the spin trap,DMPO, in bovine aortic endothelial cells via eNOS phosphorylation

Background and Purpose

Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC).

Experimental Approach

BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured.

Key Results

Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO.

Conclusion and Implications

Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.     

A role for nitroxyl (HNO) as an endothelium-derived relaxing and hyperpolarizing factor in resistance arteries

Background and purpose:

Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries.

Experimental approach:

Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording.

Key results:

Vasorelaxation to the HNO donor, Angeli''s salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K⁺ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with l-NAME, N^ω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO^•) with hydroxocobalamin (HXC) or HNO with l-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and l-cysteine reduced ACh-mediated relaxation, as did l-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with l-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO^•, as HXC and l-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP.

Conclusions and implications:

The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO^• = HNO > EDHF and EDHF > HNO = NO^• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.     

Exploring epistatic relationships of NO biosynthesis pathway genes in susceptibility to CHD

Aim:

To assess the epistatic relationships of nitric oxide (NO) biosynthesis pathway genes in susceptibility to coronary heart disease (CHD).

Methods:

A total of 2142 subjects enrolled in two case-control studies was genotyped for 7 single-nucleotide polymorphisms (SNP) within NO biosynthesis pathway genes using TaqMan assays. The association analyses were performed at both SNP and haplotype levels. Two-way SNP-SNP interactions and high-order interactions were tested using multiple unconditional logistic regression analyses and generalized multifactor dimensionality reduction (GMDR) analyses, respectively.

Results:

Two alleles (rs1049255*C and rs841*A) were identified that were significantly associated with increased risk of CHD after adjusting for all confounders (OR=1.21, 95% CI: 1.06−1.39, combined P=0.001, P_corr=0.007 and OR=1.30, 95% CI 1.12−1.50, combined P<0.001, P_corr<0.001, respectively). Significant two-way SNP–SNP interactions were found between SNP rs2297518 and these two significant polymorphisms, affecting the risk of CHD (P<0.001 for both). No significant high-order interactions were identified.

Conclusion:

The results suggested that two-way SNP–SNP interactions of polymorphisms within NO biosynthesis pathway genes contribute to CHD risk.     

The prolactin family hormones regulate vascular tone through NO and prostacyclin production in isolated rat aortic rings

Aim:

Prolactin family hormones include growth hormone, placental lactogen and prolactin, which are able to regulate angiogenesis via NO and prostaglandins. However, their effects on vascular tone are not fully understood. The aim of this study was to evaluate the effects of prolactin family hormones on rat vascular tone in vitro.

Methods:

Aortic rings were prepared from adult male rats and precontracted with phenylephrine, then treated with the hormones and drugs. The tension was measured with isometric force displacement transducer connected to a polygraph. NO production and prostacyclin release in physiological solution was determined. Cultured rat aortic endothelial cells (RAECs) were treated with the hormones and drugs, and the phosphorylation of eNOS at serine 1177 was assessed using Western bolt analysis.

Results:

Administration of growth hormone or placental lactogen (0.01–100 nmol/L) induced endothelium-dependent vasodilation. Both the hormones significantly increased the phosphorylation of eNOS in RAECs and NO level in physiological solution. Preincubation with L-NAME blocked growth hormone- or placental lactogen-induced vasodilation and NO production. Preincubation with an antibody against growth hormone receptors blocked growth hormone- and placental lactogen-induced vasodilation. Addition of a single dose of prolactin (0.01 nmol/L) induced sustained vessel relaxation, whereas multiple doses of prolactin induced a biphasic contraction-relaxation effect. The vascular effects of prolactin depended on endothelium. Prolactin significantly increased the level of prostacyclin I₂ in physiological solution. Preincubation with indomethacin or an antibody against prolactin receptors blocked prolactin-induced vasodilation.

Conclusion:

The prolactin family hormones regulate rat vascular tone, selectively promoting either relaxation or contraction of vascular smooth muscle via activation of either growth hormone receptors or prolactin receptors within the endothelium.     

Pro-ulcer effects of resveratrol in mice with indomethacin-induced gastric ulcers are reversed by l-arginine

Background and purpose:

Although resveratrol is currently being evaluated in pre-clinical studies as a potential cancer chemopreventive agent and cardiovascular stress-releasing compound, treatment with resveratrol severely delays healing of pre-existing gastric ulcers. Resveratrol treatment can also induce endothelial NOS (eNOS) expression. Here, we have attempted to modulate NO production via eNOS in order to alleviate the pro-ulcer effects of resveratrol.

Experimental approach:

Gastric ulcers were induced in mice with a single dose of indomethacin. The effects of pretreatment with l-arginine on the pro-ulcer effects of resveratrol in these mice were then assessed. We measured ulcer damage scores (DS), myeloperoxidase (MPO) activity, generation of prostaglandin E₂ (PGE₂) and NO, along with a gene expression study.

Key results:

Resveratrol significantly aggravated damage from indomethacin-induced gastric ulcers, and delayed healing, as shown by increased DS and MPO activity. The mRNA for cyclooxygenase (COX)-1, but not that for COX-2, was inhibited by resveratrol treatment, with reduced synthesis of PGE₂ by gastric tissue. However, resveratrol treatment induced eNOS gene expression and shifted the eNOS/iNOS balance. l-Arginine given before resveratrol in mice with indomethacin-induced ulcers significantly increased tissue NO synthesis and improved ulcer healing.

Conclusions and implications:

Exogenous l-arginine increased NO formation via raised levels of eNOS induced by resveratrol and protected against the pro-ulcer effects of resveratrol. Therefore, l-arginine might be useful for alleviation of the pro-ulcer side effects of resveratrol in patients.     

Inhibition of anti-IgE mediated human mast cell activation by NO donors is dependent on their NO release kinetics

Background and purpose:

Although the mast cell is a source of nitric oxide (NO), the effect of NO on human mast cells has not been defined. This study investigated if exogenous NO could affect human mast cell activation.

Experimental approach:

Effects of different NO donors on immunoglobulin E (IgE)-dependent activation of human-cultured mast cells (HCMC) derived from precursors in buffy coat were investigated by measuring histamine release. Intracellular NO in HCMC was monitored with confocal microscopy using the fluorescent NO indicator 4-amino-5-methylamino-2′, 7′-difluorofluorescein.

Key results:

Diethylamine NONOate (DEA/NO) and MAHMA NONOate (NOC-9), both have rapid NO release rates, only inhibited anti-IgE-induced histamine release when added to HCMC at the time of activation. NO donors with slower NO release kinetics were ineffective even after 30 min incubation. Confocal microscopy revealed that the effectiveness of NO donors was dependent on the availability of adequate NO inside HCMC during activation. The inhibitory action of DEA/NO was diminished by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl but potentiated by the anti-oxidant, N-acetylcysteine (NAC). Furthermore, co-incubation with NAC allowed previously ineffective NO donors to suppress HCMC activation and thus suggested that NAC could increase the availability of NO from NO donors.

Conclusions and implications:

Our results demonstrated that NO was able to modulate human mast cell activation but only when enough NO was present at the time of cell activation. Our findings explain the controversy over the effectiveness of NO on mast cell degranulation and supports the possibility that NO donors could be beneficial for treating allergic inflammation.