Plasma nitrate/nitrite levels are unchanged after long-term aerobic exercise training in older adults

Reduced nitric oxide (NO) production and bioactivity is a major contributor to endothelial dysfunction. Animal data suggest that improvements in endothelial function in response to aerobic exercise training may depend on the duration of the training program. However, no studies have examined changes in NO (as assessed by the major NO metabolites, nitrate and nitrite, NO_x) after long-term training in humans. In addition, aging may impair the ability of the vasculature to increase NO with exercise. Thus, we determined whether 24 weeks of aerobic exercise training increases plasma NO_x levels in sedentary older adults. We also examined changes in forearm blood flow (FBF) at rest and during reactive hyperemia as a measure of vasomotor function. Plasma NO_x levels were measured in 82 men and women using a modified Griess assay. FBF was assessed in a subset of individuals (n = 15) using venous occlusion plethysmography. After 24 weeks of exercise training, there were significant improvements in maximum oxygen consumption, HDL cholesterol, triglycerides, and body fat. Changes in plasma NO_x levels ranged from ?14.83 to +16.69 μmol/L; however, the mean change overall was not significant (?0.33 ± 6.30 μmol/L, p = 0.64). Changes in plasma NO_x levels were not associated with age, gender, race, HDL cholesterol, triglycerides, body weight, body fat, or maximal oxygen consumption. There were also no significant changes in basal FBF, peak FBF, hyperemic response, total hyperemic flow, or minimum forearm vascular resistance with exercise training. In conclusion, improvements in plasma NO_x levels and FBF are not evident after long-term training in older adults.     

Nitrogen dioxide oxidizes mitochondrial cytochrome c

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9–19). However, the subsequent re-evaluation of the redox potential of the NO/NO^- couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO₂^? a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3 × 10⁶ M^-1 s^-1 for the reaction of NO₂^? with ferrocytochrome c. The importance of NO₂^? was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO₂^? into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO^- or HNO) or of peroxynitrite.     

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg^?1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg^?1 iv). Plasma ET-1, nitrite/nitrate (NO_x) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO_x, significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm^?1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.     

Nitrite correlates with 3-nitrotyrosine but not with the F2-isoprostane 15(S)-8-iso-PGF2α in urine of rheumatic patients

In vitro studies suggested that nitrite may play a cytoprotective role in inflammation. The aim of the present clinical study was to investigate the relationship between the NO metabolites nitrite and nitrate and the biomarkers of oxidative stress 3-nitrotyrosine (3-NT) and 15(S)-iso-PGF_2α in patients suffering from chronic inflammatory rheumatic diseases. In morning urine from 28 patients with different chronic inflammatory rheumatic diseases (23–82 years of age) and from 41 healthy persons of both genders, nitrite and nitrate were quantitated by GC-MS, and 3-NT and 15(S)-iso-PGF_2α by GC-MS/MS. Mean creatinine-corrected urinary excretion rates of nitrite (1.1 versus 0.19 μmol/mmol, P = 0.00012) and 3-NT (1.2 versus 0.39 nmol/mmol, P = 0.01629), but not of nitrate (105 versus 106 μmol/mmol), were significantly elevated in rheumatism as compared to health. Urinary excretion rate of 15(S)-iso-PGF_2α did not differ between patients and healthy subjects (65 versus 69 pmol/mmol creatinine, P = 0.48). In rheumatism, urinary 3-NT correlated closely with nitrite (R = 0.788, P < 0.0001) and moderately with nitrate (R = 0.45, P < 0.016), but did not correlate with 15(S)-iso-PGF_2α (R = ?0.083, P = 0.68). In healthy persons there was no correlation between urinary 3-NT and nitrite or nitrate. Our study suggests that urinary nitrite may represent a novel specific biomarker of nitrative stress in chronic inflammatory rheumatic disease. In another eight patients with chronic inflammatory rheumatic diseases we found higher nitrite concentrations in synovial fluid as compared to serum (1.30 versus 0.35 μM). We hypothesize that in chronic inflammatory rheumatic diseases nitrite concentration is elevated in the inflamed joint and contributes to the inactivation of myeloperoxidase-catalyzed production of hypochloric acid by forming nitryl chloride which eventually nitrates tyrosine to form 3-NT.     

Involvement of NO-guanylyl cyclase pathway for the depression of spinal monosynaptic reflex by Mesobuthus tamulus venom in neonatal rat in vitro

AimsThe present study was undertaken to evaluate the role of nitric oxide (NO) in Mesobuthus tamulus (MBT) venom-induced depression of spinal reflexes.Main methodsExperiments were performed on isolated hemisected spinal cords from 4 to 6 day old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root.Key findingsSuperfusion of MBT venom (0.3 μg/ml) depressed the spinal reflexes in a time-dependent manner and the maximum depression was seen at 10 min (MSR by 63%; PSR by 79%). The time to produce 50% depression (T-50) of MSR and PSR was 7.7 ± 1.3 and 5.7 ± 0.5 min, respectively. Pretreatment with bicuculline (1 μM; GABA_A receptor antagonist) or strychnine (1 μM; glycine_A receptor antagonist) did not block the venom-induced depression of spinal reflexes. However, Nω-nitro-L-arginine methyl ester (L-NAME, 100 or 300 μM; NO synthase inhibitor) or hemoglobin (Hb, 100 μM; NO scavenger) antagonized the venom-induced depression of MSR. Further, soluble guanylyl cylase inhibitors (1 H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ; 1 μM or methylene blue, 100 μM) also antagonized the venom-induced depression of MSR but not PSR. Nitrite concentration (indicator of NO activity) of the cords exposed to venom (0.3 μg/ml) was not different from the control group.SignificanceThe results indicate that venom-induced depression of MSR is mediated via NO-guanylyl cyclase pathway without involving GABAergic or glycinergic system.     

Effect of dietary conjugated linoleic acid on serum levels of N2O5 and l-citrulline in goat kids

Forty new born male kids were allotted into two groups for the evaluation of the effects of dietary conjugated linoleic acid (CLA) on nitric oxide (NO) and l-citrulline levels in serum. The control group received no supplement CLA and the CLA group received 20 g/kg milk DM of CLA from birth to 1-month old. The kids were fed colostrum for 2 days and milk replacer from days 3 to 29. Blood samples (n = 40) were taken at 1, 8, 15, 22, and 29 days of age. N₂O₅ (nitrite + nitrate) concentration in serum ranged from 21.93 to 26.15 and 34.90 to 40.59 μmol in control and CLA kids. The l-citrulline values ranged from 0.30 to 0.40 and 15.54 to 19.81 μmol in control and CLA kids.     

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: role of α7 nicotinic cholinergic receptor/eNOS signaling

AimsWe recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect.Main methodsNicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE₂) rats.Key findingsNicotine infusion (5 × 10^? 5, 1 × 10^? 4, and 5 × 10^? 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N^G-nitro-l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E₂ in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α₇ nicotinic cholinergic receptor (α₇ nAChRs) were significantly higher in renal tissues of OVXE₂ compared with OVX rats, suggesting a facilitatory effect for E₂ on α₇ nAChRs/eNOS signaling.SignificanceEstrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α₇ nAChRs in this estrogen-dependent phenomenon.     

Low serum zinc levels in patients undergoing coronary angiography correlate with immune activation and inflammation

IntroductionLow serum zinc concentrations are associated with adverse outcomes. To explain this phenomenon we aimed to investigate whether low zinc levels are related to immune activation, renal function and coronary artery disease (CAD).MethodsSerum concentrations of zinc and the immune activation markers neopterin and C-reactive protein (CRP) were measured in 2048 patients derived from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study, a cohort study among patients referred for coronary angiography.ResultsZinc concentrations did not differ between patients with CAD (mean ± SD: 13.3 ± 2.4 μmol/L) and controls (13.3 ± 2.2 μmol/L; Welch's t test: p = n.s.) but CAD patients had higher neopterin (8.6 ± 7.4 nmol/L) and CRP (9.7 ± 19.6 mg/L) concentrations compared to controls (neopterin: 7.5 ± 4.8 nmol/L, p = 0.0005; CRP: 5.5 ± 10.0 mg/L, p < 0.0001). There was an inverse correlation between serum zinc concentrations and neopterin (Spearman's rank correlation: r_s = ?0.222) and CRP (r_s = ?0.166; both p < 0.0001) concentrations.ConclusionsOur results indicate increased inflammatory processes in patients with low zinc levels. Further studies should clarify whether inflammation related processes such as renal wasting contribute to zinc deficiency and underlie the adverse health consequences of low serum zinc levels.     

Ouabain stimulates atrial natriuretic peptide secretion via the endothelin-1/ET(B) receptor-mediated pathway in beating rabbit atria

AimsOuabain has been reported to increase the secretion of atrial natriuretic peptide (ANP) in vitro. However, the mechanism by which ouabain increases ANP secretion is not well known. Therefore, the purpose of the present study was to investigate the underlying mechanism of ouabain-stimulated ANP secretion.Main methodsA perfused beating rabbit atrial model was used. The ANP and ET-1 levels in the atrial perfusates were measured by radioimmunoassays.Key findingsOuabain (1.0, 3.0 and 6.0 μmol/L) significantly increased atrial ANP secretion in a dose-dependent manner, while the endothelin (ET)-1 levels were increased by the higher doses (3.0 and 6.0 μmol/L) of ouabain. Ouabain-increased atrial ET-1 release was blocked by PD98059 (30.0 μmol/L), an inhibitor of mitogen-activated protein kinase (MAPK). Nifedipine (1.0 μmol/L), an inhibitor of L-type Ca²⁺ channels, completely abolished ouabain-increased ANP secretion without changing the ouabain-induced atrial dynamics. KB-R7943 (3.0 μmol/L), an inhibitor of Na⁺–Ca²⁺ exchangers, completely blocked the effects of ouabain-increased atrial dynamics, but did not modulate ouabain-increased ANP secretion. ET-1 significantly stimulated atrial ANP release in a dose-dependent manner. The effects of ET-1 and ouabain on ANP secretion were completely blocked by BQ788 (0.3 μmol/L), an inhibitor of ET-1 type B (ET_B) receptors, but not by BQ123 (0.3 μM), an inhibitor of ET-1 type A receptors. Ouabain-increased atrial ANP secretion was blocked by PD98059 and indomethacin (30.0 μmol/L), an inhibitor of cyclooxygenase.SignificanceOuabain significantly stimulated atrial ANP secretion via an ET-1-ET_B receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation.     

Cholesterol metabolism differs after statin therapy according to the type of hyperlipemia

AimNon-cholesterol sterols reflect cholesterol metabolism. Statins reduce cholesterol synthesis usually with a rise in cholesterol absorption. Common hyperlipemias have shown different patterns of cholesterol metabolism. We evaluated whether cholesterol absorption and synthesis may differ after statin therapy in primary hyperlipemias.Main methodsWe determined lipid profile, apoprotein B and serum sterols (lathosterol, sitosterol, campesterol by gas chromatography/mass spectrometry) before and after statins in 80 untreated hyperlipemic patients, 40 with polygenic hypercholesterolemia (PH) and 40 with familial combined hyperlipemia (FCH).Key findingsAt baseline in FCH lathosterol was significantly higher while campesterol and sitosterol were significantly lower than in PH. After statins, the reduction in LDL-C did not significantly differ between the two groups; in PH there was a significant decrease of lathosterol from 96.1 to 52.6 102 μmol/mmol cholesterol (p = 0.0001) with no significant modifications in campesterol and sitosterol; on the opposite, in FCH lathosterol decreased from 117 to 43 102 μmol/mmol cholesterol (p = 0.0001) and campesterol and sitosterol significantly increased from 38 to 48 102 μmol/mmol cholesterol (p = 0.0001), and from 75 to 86 102 μmol/mmol cholesterol, (p = 0.022), respectively. After statin therapy only in FCH Δ-LDL-C showed a significant inverse correlation with Δ-sitosterol and with Δ-campesterol.SignificancePrimary hyperlipemias show different patterns of response to statins: in PH LDL reduction appears completely “synthesis inhibition” dependent, while in FCH LDL decrease appears to be synthesis dependent, partially limited by absorption increase. Studying cholesterol metabolism before and after hypolipemic therapy might be useful in identifying the best tailored treatment.     

Physiological recycling of endogenous nitrate by oral bacteria regulates gastric mucus thickness

BackgroundInorganic nitrate from exogenous and endogenous sources is accumulated in saliva, reduced to nitrite by oral bacteria and further converted to nitric oxide (NO) and other bioactive nitrogen oxides in the acidic gastric lumen. To further explore the role of oral microbiota in this process we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite.MethodsMice were given either nitrate (100 mg/kg/d) or nitrite (0.55–11 mg/kg/d) in the drinking water for 7 days, with the lowest nitrite dose resembling the levels provided by swallowing of fasting saliva. The gastric mucus layer was measured in vivo.ResultsGF animals were almost devoid of the firmly adherent mucus layer compared to conventional mice. Dietary nitrate increased the mucus thickness in conventional animals but had no effect in GF mice. In contrast, nitrite at all doses, restored the mucus thickness in GF mice to the same levels as in conventional animals. The nitrite-mediated increase in gastric mucus thickness was not inhibited by the soluble guanylyl cyclase inhibitor ODQ. Mice treated with antibiotics had significantly thinner mucus than controls. Additional studies on mucin gene expression demonstrated down regulation of Muc5ac and Muc6 in germ free mice after nitrite treatment.ConclusionOral bacteria remotely modulate gastric mucus generation via bioactivation of salivary nitrate. In the absence of a dietary nitrate intake, salivary nitrate originates mainly from NO synthase. Thus, oxidized NO from the endothelium and elsewhere is recycled to regulate gastric mucus homeostasis.     

Effects of oral administration of (L)-arginine, (L)-NAME and allopurinol on intestinal ischemia/reperfusion injury in rats

AimsIntestinal ischemia/reperfusion (I/R) injury is implicated in many clinical conditions, and it performs a fundamental role in their pathophysiologies. Oral administration of antioxidants and nitric oxide (NO) donors ameliorate intestinal injury. Here, the effects of l-arginine, allopurinol and N^G-nitro-l-arginine methyl ester (l-NAME) were investigated.Main methodsOne hundred twenty-eight male Wistar rats were separated into 4 groups and subjected to occlusion of the superior mesenteric artery for 60 min. The Control group did not receive any substance before the surgical operation. However, the 3 other groups received the following: l-arginine (800 mg/kg body weight; l-Arg group), l-NAME (50 mg/kg; l-NAME group) or allopurinol (100 mg/kg; Allo group). Each substance was given by mouth in 3 equal doses 24, 12 and 1 h before the surgical operation. Each group was then divided into 4 subgroups, which underwent different durations of reperfusion (0, 1, 8 or 24 h). At the end of each time point, blood and tissue samples were collected, and histological examinations were performed. Serum nitrite and catalase, intestinal tissue myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) levels were determined.Key findingsAt each reperfusion time point, the Allo group exhibited the mildest histological lesions in contrast to the l-NAME group, which showed the most severe lesions. MPO was decreased significantly in the Allo and l-Arg groups during reperfusion, and allopurinol administration caused earlier and stronger effect. iNOS and NT levels were higher in the l-Arg group and lower in the Allo group. Serum nitrite and catalase were increased in the l-NAME group after 24 h.SignificanceOral administration of allopurinol exerted a strong and protective effect on the intestinal tissue that was subjected to I/R earlier than l-arginine. This finding was also supported with the MPO, iNOS and NT data.     

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5 s), therefore direct NO quantification is challenging. An indirect method – based on quantifying conversion of an l-Arg- to l-Cit-derivative by ¹H NMR spectroscopy – is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a–j and 11a–c were better or equivalent substrates for the eNOS enzyme (NO₂^? production = 19–46 μM) than native l-Arg (NO₂^? production = 25 μM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by ¹H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.     

Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats

AimsThe present study aims to elucidate the role of oxidative stress in erectile dysfunction associated with aging and to investigate the effect of treatment with vitamin E in this respect.Main methodsRats were divided into four groups: young (3-month-old), aged rats (18-month-old), aged rats given 80 IU of vitamin E/rat/day for 21-days, aged rats given 5 mg/kg of sildenafil/day for 21-days. Intracavernosal pressure/mean arterial pressure (ICP/MAP), nitric oxide production, TBARS, GSH levels and SOD activity in corpus cavernosum were measured.Key findingsSignificant decrease in ICP/MAP was observed in aged rats at both low and high frequency of stimulation. Significant increase in ICP/MAP was observed in aged rats treated with vitamin E over the range of 0.8 to 5 Hz but young control values were not restored. Percentage potentiation of ICP/MAP than aged group at 0.8 Hz was 326 ± 41.3% and 897 ± 72.2% for vitamin E and sildenafil respectively. Decreased levels of NO₂/NO₃ and SOD activity in the penile tissue observed with aging were elevated back to control by either vitamin E or sildenafil. Penile concentration of TBARS was 20.86 ± 0.83 for aged rats vs. 11.39 ± 0.79 nmol/g tissue for young controls. Both vitamin E and sildenafil reduced penile TBARS in aged rats.SignificanceThis study proves that antioxidant therapy with vitamin E ameliorates the age-associated erectile dysfunction. Sildenafil may exert some antioxidant properties which add to the advantages of its long-term use. The effect of combinations of low-dose sildenafil and vitamin E on age-associated erectile dysfunction merits to be studied.     

Nitroxyl (HNO) suppresses vascular Nox2 oxidase activity

Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli’s salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1 μmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli’s salt (1 μmol/L) or IPA/NO (1 μmol/L). Similarly, phorbyl 12,13-dibutyrate (10 μmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1 μmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3 mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100 μmol/L; NO scavenger), ODQ (10 μmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10 μmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2^−/y) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1 μmol/L) had no effect on superoxide levels in arteries from Nox2^−/y mice. Finally, angiotensin II (1–1000 μmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1 μmol/L), whereas constrictor responses to either the thromboxane A₂ mimetic U46619 (1–100 nmol/L) or high potassium (122.7 mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC–cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.     

Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: role of phenolic hydroxyl groups in the resorcinol moiety

AimsIn this study, we examined the inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity.Main methodsDiltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined.Key findingsAmong the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC₅₀ = 11.7 and 1.65 μM, respectively). The IC₅₀ values of Δ⁹-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ⁹-THC, CBD, and CBN inhibited the activity to a similar extent (IC₅₀ = 23–31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K_i = 1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K_i = 12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety.SignificanceThis study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.     

In vitro antifungal activity of ajoene on five clinical isolates of Histoplasma capsulatum var. capsulatum

BackgroundThe number of histoplasmosis cases have considerably increased since the advent of AIDS, and the therapy for this mycosis is not always effective, as well as having adverse effects.AimsTo evaluate the inhibitory effect of ajoene on five clinical isolates of Histoplasma capsulatum, on the mycelial form, using Sabouraud dextrose broth (SDB) and RPMI-1640 culture media.MethodsGrowth curves and inhibitory activity of the drug (at concentrations of 1.25 ug/ml to 20 μg/ml) were performed at room temperature, under mechanical agitation, and the turbidimetric readings (540 nm) were recorded every 48 h for 14 days, in both culture media. Generation times (GT) were calculated and graphs were constructed to estimate Minimal Inhibitory Concentrations (MIC) and Inhibitory Concentration 50% (IC₅₀). The fungicidal minimal concentrations (FMC) were determined by plate cultures. The U-Mann-Whitney and t-test with a significance level of 0.05 were used to evaluate the statistical significance between culture media and GT, MIC, IC₅₀ MFC and fungistatic effect (FE).ResultsIn both media and for all isolates, growth curves showed a GT of 43 to 67 hrs, an FE at 1.25-2.5 μg/ml, and a MFC at 5-10 μg/ml of ajoene. Values of MIC were 2.5-5 in SDB and in RPMI medium these values were 1.25-5 μg/ml of ajoene. For IC₅₀, in SDB, the values were 1.9-2.6 ug/ml and in RPMI medium, they were of 3.8-4.3 μg/ml of ajoene. There were no significance differences between culture media for GT, FE, MIC, IC₅₀ and MFC (p > 0.05).ConclusionsThese findings corroborate that ajoene inhibits the growth of the mycelial form of H. capsulatum.     

Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins

The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep?, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), β-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (K_D > 1 μM). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with K_D values 0.14 ± 0.02 μM and 0.17 ± 0.02 μM, respectively. StARD3 expressed a relative high specificity towards lutein with a K_D value of 0.59 ± 0.03 μM, whereas StARD1 exhibited a relatively low selectivity and affinity (K_D > 1 μM) towards the various carotenoids tested.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (K_ATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC).Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30 min of occlusion of the left coronary artery and 120 min of reperfusion with or without IPC.Key findingsIPC (1 cycle), OXT (0.03 μg/kg, i.p.) or IPC + OXT yield significant infarct size reductions (21.8 ± 1.5%, 20.5 ± 1.2% and 19.4 ± 1.4%, respectively, versus 38.9 ± 3.5% in the S-CONT group; P < 0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P < 0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P < 0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P < 0.005, P < 0.01 and P < 0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial K_ATP channels and NO. An iNOS inhibitor (1400 W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC + OXT and 1400 W + OXT interventions significantly preserved ATP levels in the heart.SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.