Complete autonomic blockade reveals nitric oxide contribution to blood pressure regulation in obese Black women.

PURPOSE: Hypertension is one of the major causes of cardiovascular morbidity and mortality in the USA and disproportionately affects Black women. Endothelial-derived nitric oxide (eNO) substantially regulates blood pressure in humans, and impaired NO-mediated vasodilation has been reported in the Black population. Previous studies using an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA) did not fully determine the NO contribution to blood pressure because of baroreflex buffering. Therefore, in the present study we used trimethaphan, a ganglionic blocker, to inhibit baroreflex buffering and study NO modulation of blood pressure in Black women during L-NMMA infusion. METHODS: L-NMMA at doses of 250 µg/kg per minute was infused in combination with trimethaphan at doses of 4 mg/min to eliminate baroreflex mechanisms. Heart rate (HR) was obtained with continuous electrocardiogram monitoring, and continuous blood pressure was measured with the volume clamp method. The increase in systolic blood pressure (SBP) during both infusions was used to estimate the contribution of NO to blood pressure. RESULTS: Ten Black (age range 30-50 years, body mass index [BMI] 30-45 kg/m2), and nine White women (age range 30-50 years, body mass index 30-45 kg/m2) were enrolled in this study. During autonomic blockade, there was no difference in the decrease in SBP between Black and White women (- 20 ± 16.45 vs. - 24 ± 15.49 mm Hg, respectively; P = 0.659). When autonomic blockade was combined with L-NMMA, Black women had a significant increase in SBP compared to White women (54 ± 13.62 vs. 39 ± 09.64 mm Hg, respectively; P = 0.022, respectively). CONCLUSION: Autonomic blood pressure regulation was similar between Black and White women. However, NO contribution to blood pressure was significantly greater in Black women compared to White women. REGISTRATION: ClinicalTrials.gov: NCT01122407.

Nitric oxide contributes to cerebrovascular shear-mediated dilatation but not steady-state cerebrovascular reactivity to carbon dioxide.

Cerebrovascular CO2 reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated dilatation (cSMD) that may better reflect endothelial function. We aimed to determine the nitric oxide (NO)-dependency of CVR and cSMD. Eleven volunteers underwent a steady-state CVR test and transient CO2 test of cSMD during intravenous infusion of the NO synthase inhibitor NG -monomethyl-l-arginine (l-NMMA) or volume-matched saline (placebo; single-blinded and counter-balanced). We measured cerebral blood flow (CBF; duplex ultrasound), intra-arterial blood pressure and PaCO2${P_{{\rm{aC}}{{\rm{O}}_{\rm{2}}}}}$ . Paired arterial and jugular venous blood sampling allowed for the determination of trans-cerebral NO2- exchange (ozone-based chemiluminescence). l-NMMA reduced arterial NO2- by ∼25% versus saline (74.3 ± 39.9 vs. 98.1 ± 34.2 nM; P = 0.03). The steady-state CVR (20.1 ± 11.6 nM/min at baseline vs. 3.2 ± 16.7 nM/min at +9 mmHg PaCO2${P_{{\rm{aC}}{{\rm{O}}_{\rm{2}}}}}$ ; P = 0.017) and transient cSMD tests (3.4 ± 5.9 nM/min at baseline vs. -1.8 ± 8.2 nM/min at 120 s post-CO2 ; P = 0.044) shifted trans-cerebral NO2- exchange towards a greater net release (a negative value indicates release). Although this trans-cerebral NO2- release was abolished by l-NMMA, CVR did not differ between the saline and l-NMMA trials (57.2 ± 14.6 vs. 54.1 ± 12.1 ml/min/mmHg; P = 0.49), nor did l-NMMA impact peak internal carotid artery dilatation during the steady-state CVR test (6.2 ± 4.5 vs. 6.2 ± 5.0% dilatation; P = 0.960). However, l-NMMA reduced cSMD by ∼37% compared to saline (2.91 ± 1.38 vs. 4.65 ± 2.50%; P = 0.009). Our findings indicate that NO is not an obligatory regulator of steady-state CVR. Further, our novel transient CO2 test of cSMD is largely NO-dependent and provides an in vivo bioassay of NO-mediated cerebrovascular function in humans. KEY POINTS: Emerging evidence indicates that a transient CO2 stimulus elicits shear-mediated dilatation of the internal carotid artery, termed cerebral shear-mediated dilatation. Whether or not cerebrovascular reactivity to a steady-state CO2 stimulus is NO-dependent remains unclear in humans. During both a steady-state cerebrovascular reactivity test and a transient CO2 test of cerebral shear-mediated dilatation, trans-cerebral nitrite exchange shifted towards a net release indicating cerebrovascular NO production; this response was not evident following intravenous infusion of the non-selective NO synthase inhibitor NG -monomethyl-l-arginine. NO synthase blockade did not alter cerebrovascular reactivity in the steady-state CO2 test; however, cerebral shear-mediated dilatation following a transient CO2 stimulus was reduced by ∼37% following intravenous infusion of NG -monomethyl-l-arginine. NO is not obligatory for cerebrovascular reactivity to CO2 , but is a key contributor to cerebral shear-mediated dilatation.

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Effect of NG -monomethyl l-arginine on microvascular blood flow and glucose metabolism after an oral glucose load.

OBJECTIVE: The aim of this study was to investigate whether the effects on local blood flow and metabolic changes observed in the skin after an endogenous systemic increase in insulin are mediated by the endothelial nitric oxide pathway, by administering the nitric oxide synthase inhibitor NG -monomethyl l-arginine using microdialysis. METHODS: Microdialysis catheters, perfused with NG -monomethyl l-arginine and with a control solution, were inserted intracutaneously in 12 human subjects, who received an oral glucose load to induce a systemic hyperinsulinemia. During microdialysis, the local blood flow was measured by urea clearance and by laser speckle contrast imaging, and glucose metabolites were measured. RESULTS: After oral glucose intake, microvascular blood flow and glucose metabolism were both significantly suppressed in the NG -monomethyl l-arginine catheter compared to the control catheter (urea clearance: P < .006, glucose dialysate concentration: P < .035). No significant effect of NG -monomethyl l-arginine on microvascular blood flow was observed with laser speckle contrast imaging (P = .81). CONCLUSION: Local delivery of NG -monomethyl l-arginine to the skin by microdialysis reduces microvascular blood flow and glucose delivery in the skin after oral glucose intake, presumably by decreasing local insulin-mediated vasodilation.

Nitric oxide synthase inhibition with N(G)-monomethyl-l-arginine: Determining the window of effect in the human vasculature.

Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the window of effect for L-NMMA on human vascular function is unknown, which is critical for designing and interpreting human-based studies. This study utilized the passive leg movement (PLM) assessment of vascular function, which is predominantly NO-mediated, in 7 young male subjects under control conditions, immediately following intra-arterial L-NMMA infusion (0.24 mg⋅dl-1⋅min-1), and at 45-60 and 90-105 min post L-NMMA infusion. The leg blood flow (LBF) and leg vascular conductance (LVC) responses to PLM, measured with Doppler ultrasound and expressed as the change from baseline to peak (ΔLBFpeak and ΔLVCpeak) and area under the curve (LBFAUC and LVCACU), were assessed. PLM-induced robust control ΔLBFpeak (1135 ± 324 ml⋅min-1) and ΔLVCpeak (10.7 ± 3.6 ml⋅min-1⋅mmHg-1) responses that were significantly attenuated (704 ± 196 ml⋅min-1 and 6.7 ± 2 ml⋅min-1⋅mmHg-1) immediately following L-NMMA infusion. Likewise, control condition PLM ΔLBFAUC (455 ± 202 ml) and ΔLVCAUC (4.0 ± 1.4 ml⋅mmHg-1) were significantly attenuated (141 ± 130 ml and 1.3 ± 1.2 ml⋅mmHg-1) immediately following L-NMMA infusion. However, by 45-60 min post L-NMMA infusion all PLM variables were not significantly different from control, and this was still the case at 90-105 min post L-NMMA infusion. These findings reveal that the potent reduction in NO bioavailability afforded by NOS inhibition with L-NMMA has a window of effect of less than 45-60 min in the human vasculature. These data are particularly important for the commonly employed approach of pharmacologically inhibiting NOS with L-NMMA in the human vasculature.

Normal increases in insulin-stimulated glucose uptake after ex vivo contraction in neuronal nitric oxide synthase mu (nNOSµ) knockout mice.

Nitric oxide (NO) is involved in skeletal muscle glucose uptake during exercise and also in the increase in insulin sensitivity after exercise. Given that neuronal nitric oxide synthase (NOS) isoform mu (nNOSµ) is a major isoform of NOS in skeletal muscle, we examined if the increase in skeletal muscle insulin-stimulated glucose uptake 3.5 h following ex vivo contraction of extensor digitorum longus (EDL) is reduced in muscles from nNOSµ+/- and nNOSµ-/- mice compared with nNOSµ+/+ mice. 3.5 h post-contraction/basal, muscles were exposed to saline or insulin (120µU/ml) with or without the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) during the last 30 min and glucose uptake was determined by radioactive tracers. Skeletal muscle insulin-stimulated glucose uptake from nNOSµ+/+, nNOSµ+/-, and nNOSµ-/- mice increased approximately twofold 3.5 h following ex vivo contraction when compared to rest. L-NMMA significantly attenuated this increase in muscle insulin-stimulated glucose uptake by around 50%, irrespective of genotype. Low levels of NOS activity were detected in muscles from nNOSµ-/- mice. In conclusion, NO mediates increases in mouse skeletal muscle insulin response following ex vivo contraction independently of nNOSµ.

Assessment of real-time and quantitative changes in renal hemodynamics in healthy overweight males: Contrast-enhanced ultrasonography vs para-aminohippuric acid clearance.

OBJECTIVE: To determine the ability of renal contrast-enhanced ultrasonography (CEUS) to detect acute drug-induced changes in renal perfusion (using the glucagon-like peptide (GLP)-1 receptor agonist exenatide and nitric oxide [NO]-synthase inhibitor L-NG -monomethyl arginine [l-NMMA]), and assess its correlation with gold standard-measured effective renal plasma flow in humans. METHODS: In this prespecified exploratory analysis of a placebo-controlled cross-over study, renal hemodynamics was assessed in 10 healthy overweight males (aged 20-27 years; BMI 26-31 kg/m2 ) over two separate testing days; during placebo (isotonic saline) and subsequent exenatide infusion (Day-A), and during l-NMMA, and subsequent exenatide plus l-NMMA infusion (Day-B). Renal cortical microvascular blood flow was estimated following microbubble infusion and CEUS destruction-refilling-sequences. Renal cortical microvascular blood flow was compared with simultaneously measured effective renal plasma flow in humans, derived from para-aminohippuric acid-clearance methodology. RESULTS: On Day-A, effective renal plasma flow increased by 68 [26-197] mL/min/1.73 m2 during exenatide vs placebo infusion (+17%; P = .015). In parallel, exenatide increased renal cortical microvascular blood flow, from 2.42 × 10-4 [6.54 × 10-5 -4.66 × 10-4 ] AU to 4.65 × 10-4 [2.96 × 10-4 -7.74 × 10-4 ] AU (+92%; P = .027). On Day-B, effective renal plasma flow and renal cortical microvascular blood flow were reduced by l-NMMA, with no significant effect of concomitant exenatide on renal hemodynamic-indices assessed by either technique. Effective renal plasma flow correlated with renal cortical microvascular blood flow on Day-A (r = .533; P = .027); no correlation was found on Day-B. CONCLUSIONS: Contrast-enhanced ultrasonography can detect acute drug-induced changes human renal hemodynamics. CEUS-assessed renal cortical microvascular blood flow moderately associates with effective renal plasma flow, particularly when perfusion is in normal-to-high range. Renal CEUS cannot replace effective renal plasma flow measurements, but may be a complementary tool to characterize regional kidney perfusion.

Nitric oxide is required for the insulin sensitizing effects of contraction in mouse skeletal muscle.

KEY POINTS: People with insulin resistance or type 2 diabetes can substantially increase their skeletal muscle glucose uptake during exercise and insulin sensitivity after exercise. Skeletal muscle nitric oxide (NO) is important for glucose uptake during exercise, although how prior exercise increases insulin sensitivity is unclear. In the present study, we examined whether NO is necessary for normal increases in skeletal muscle insulin sensitivity after contraction ex vivo in mouse muscle. The present study uncovers, for the first time, a novel role for NO in the insulin sensitizing effects of ex vivo contraction, which is independent of blood flow. ABSTRACT: The factors regulating the increase in skeletal muscle insulin sensitivity after exercise are unclear. We examined whether nitric oxide (NO) is required for the increase in insulin sensitivity after ex vivo contractions. Isolated C57BL/6J mouse EDL muscles were contracted for 10 min or remained at rest (basal) with or without the NO synthase (NOS) inhibition (NG -monomethyl-l-arginine; l-NMMA; 100 µm). Then, 3.5 h post contraction/basal, muscles were exposed to saline or insulin (120 µU ml-1 ) with or without l-NMMA during the last 30 min. l-NMMA had no effect on basal skeletal muscle glucose uptake. The increase in muscle glucose uptake with insulin (57%) was significantly (P < 0.05) greater after prior contraction (140% increase). NOS inhibition during the contractions had no effect on this insulin-sensitizing effect of contraction, whereas NOS inhibition during insulin prevented the increase in skeletal muscle insulin sensitivity post-contraction. Soluble guanylate cyclase inhibition, protein kinase G (PKG) inhibition or cyclic nucleotide phosphodiesterase inhibition each had no effect on the insulin-sensitizing effect of prior contraction. In conclusion, NO is required for increases in insulin sensitivity several hours after contraction of mouse skeletal muscle via a cGMP/PKG independent pathway.

Prolonged exposure of resveratrol induces reactive superoxide species-independent apoptosis in murine prostate cells.

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.

l-arginine and l-NMMA for assessing cerebral endothelial dysfunction in ischaemic cerebrovascular disease: A systematic review.

Endothelial dysfunction (ED), in particular cerebral ED, may be an essential biomarker for ischaemic cerebrovascular disease. However, there is no consensus on methods to best estimate cerebral ED. In this systematic review, we evaluate the use of l-arginine and NG -monomethyl-l-arginine (l-NMMA) for assessment of cerebral ED. A systematic search of PubMed, EMBASE and the Cochrane Library was done. We included studies investigating cerebrovascular response to l-arginine or l-NMMA in human subjects with vascular risk factors or ischaemic cerebrovascular disease. Seven studies (315 subjects) were eligible according to inclusion and exclusion criteria. Studies investigated the effect of age (n=2), type 2 diabetes mellitus (DM) (n=1), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (n=1), leukoaraiosis (n=1), and prior ischaemic stroke or transient ischaemic attack (TIA) (n=2) on cerebral ED. Most studies applied transcranial Doppler to quantify cerebral ED. Endothelium-dependent vasodilatation (EDV) induced by l-arginine was impaired in elderly and subjects with leukoaraiosis, but enhanced in CADASIL patients. Studies including subjects with prior ischaemic stroke or TIA reported both enhanced and impaired EDV to l-arginine. Responses to l-NMMA deviated between subjects with type 2 DM and the elderly. We found only few studies investigating cerebral endothelial responses to l-arginine and l-NMMA in subjects with vascular risk factors or ischaemic cerebrovascular disease. Inconsistencies in results were most likely due to variations in methods and included subject populations. In order to use cerebral ED as a prognostic marker, further studies are required to evaluate the association to cerebrovascular disease.

Ascorbic acid improves brachial artery vasodilation during progressive handgrip exercise in the elderly through a nitric oxide-mediated mechanism.

The proposed mechanistic link between the age-related attenuation in vascular function and free radicals is an attractive hypothesis; however, direct evidence of free radical attenuation and a concomitant improvement in vascular function in the elderly is lacking. Therefore, this study sought to test the hypothesis that ascorbic acid (AA), administered intra-arterially during progressive handgrip exercise, improves brachial artery (BA) vasodilation in a nitric oxide (NO)-dependent manner, by mitigating free radical production. BA vasodilation (Doppler ultrasound) and free radical outflow [electron paramagnetic resonance (EPR) spectroscopy] were measured in seven healthy older adults (69 ± 2 yr) during handgrip exercise at 3, 6, 9, and 12 kg (∼13-52% of maximal voluntary contraction) during the control condition and nitric oxide synthase (NOS) inhibition via N(G)-monomethyl-L-arginine (L-NMMA), AA, and coinfusion of l-NMMA + AA. Baseline BA diameter was not altered by any of the treatments, while L-NMMA and L-NMMA + AA diminished baseline BA blood flow and shear rate. AA improved BA dilation compared with control at 9 kg (control: 6.5 ± 2.2%, AA: 10.9 ± 2.5%, P = 0.01) and 12 kg (control: 9.5 ± 2.7%, AA: 15.9 ± 3.7%, P < 0.01). NOS inhibition blunted BA vasodilation compared with control and when combined with AA eliminated the AA-induced improvement in BA vasodilation. Free radical outflow increased with exercise intensity but, interestingly, was not attenuated by AA. Collectively, these results indicate that AA improves BA vasodilation in the elderly during handgrip exercise through an NO-dependent mechanism; however, this improvement appears not to be the direct consequence of attenuated free radical outflow from the forearm.

Sex-related differences in reactivity of cerebral arterioles during moderate exercise training.

OBJECTIVE: Our goals were to determine the influence of sex on reactivity of cerebral arterioles and whether MExT could influence sex-related differences in reactivity of cerebral arterioles. MATERIALS AND METHODS: Responses of cerebral arterioles were measured in Sed and MExT adult male and female Sprague-Dawley rats to eNOS-dependent (ADP), nNOS-dependent (NMDA), and NOS-independent (nitroglycerin) agonists before and following L-NMMA. In addition, protein expression for eNOS and nNOS was determined. RESULTS: NOS-dependent vasodilation was enhanced in Sed and MExT female rats compared to their male counterparts. L-NMMA produced a greater decrease in baseline diameter of arterioles in females compared to males, and produced less inhibition of NOS-dependent vasodilation in females. Expression of eNOS protein was significantly increased in Sed female when compared to Sed male rats; nNOS protein was similar in Sed males and females, but increased in MExT females. CONCLUSIONS: The findings from this study indicate that while NOS-dependent vascular reactivity is increased in females, MExT does not alter vasodilation in males or females. These studies provide insights into the influence of sex and MExT on the cerebral microcirculation and may have implications regarding mechanisms that protect the brain in females compared to males.

Acute renal haemodynamic effects of glucagon-like peptide-1 receptor agonist exenatide in healthy overweight men.

AIMS: To determine the acute effect of glucagon-like peptide-1 (GLP-1) receptor agonist exenatide and the involvement of nitric oxide (NO) on renal haemodynamics and tubular function, in healthy overweight men. METHODS: Renal haemodynamics and tubular electrolyte handling were measured in 10 healthy overweight men (aged 20-27 years; BMI 26-31 kg/m(2)) during intravenous administration of placebo (saline 0.9%), exenatide, and exenatide combined with the NO-synthase inhibitor L-N(G)-monomethyl arginine (L-NMMA). Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by inulin and para-aminohippurate clearance techniques, respectively, based on timed urine sampling. Glomerular hydrostatic pressure and vascular resistance of afferent and efferent renal arterioles were calculated using the Gomez formulae. Urinary electrolytes, osmolality and pH were also measured. RESULTS: GFR increased by a mean of 18 ± 20 (+20%; p = 0.021) and ERPF increased by a median (interquartile range) of 68 (26; 197) ml/min/1.73 m(2) (+14%; p = 0.015) during exenatide infusion versus placebo. During L-NMMA infusion, exenatide increased GFR by mean 8 ± 12 ml/min/1.73 m(2) (+9%; p = 0.049). Exenatide increased estimated glomerular pressure by +6% (p = 0.015) and reduced afferent renal vascular resistance by -33% (p = 0.038), whereas these effects were blunted during L-NMMA infusion. Exenatide increased absolute and fractional sodium excretion, urinary osmolality and urinary pH. The tubular effects of exenatide were not altered by concomitant L-NMMA infusion. CONCLUSIONS: Exenatide infusion in healthy overweight men acutely increases GFR, ERPF and glomerular pressure, probably by reducing afferent renal vascular resistance, and at least partially in an NO-dependent manner. As baseline renal haemodynamics in patients with type 2 diabetes differ from those in healthy individuals, clinical studies on the renal effects of GLP-1 receptor agonists are warranted.

The diameter response of retinal arterioles in diabetic maculopathy is reduced during hypoxia and is unaffected by the inhibition of cyclo-oxygenase and nitric oxide synthesis.

PURPOSE: Diabetic retinopathy is accompanied with changes in the diameter regulation and oxygenation of retinal vessels. Previous studies have shown that in normal persons and in diabetic patients without retinopathy hypoxia-induced vasodilatation is mediated by cyclo-oxygenase (COX) products and nitric oxide (NO). The purpose of the present study was to study whether these effects can be reproduced in patients with diabetic maculopathy. METHODS: Eighteen patients with diabetic maculopathy aged 29-57 years were examined using the Dynamic Vessel Analyzer. The resting diameter and the diameter changes of retinal arterioles during isometric exercise and flicker stimulation were studied before and during breathing a hypoxic gas mixture. The examinations were also performed before and during intravenous infusion of the NOS inhibitor L-NMMA, and were repeated on a second day after topical administration of the COX-inhibitor diclofenac. RESULTS: The diameter of retinal arterioles showed no significant change during hypoxia or L-NMMA infusion, or after topical application of diclofenac (p > 0.25 for all comparisons). The resting diameter of the venules was significantly increased during hypoxia (p = 0.003) and decreased during L-NMMA infusion (p < 0.0001). The diameter of retinal venules during isometric exercise increased significantly during hypoxia (p = 0.01). Flicker stimulation induced significant dilatation of the venules, which was significantly reduced during hypoxia and increased during L-NMMA infusion (p < 0.0001 for all comparisons). CONCLUSION: Hypoxia-induced dilatation of retinal arterioles is severely reduced in patients with diabetic maculopathy. Future intervention studies aimed at normalizing the diameter regulation of retinal arterioles in diabetic patients should preferentially be conducted in the early stages of the disease where the potential for changing the vessel diameter is preserved. ClinicalTrials.gov identifier: NCT01689090.

Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.

N(G) ,N(G) -dimethyl-l-arginine (asymmetric dimethylarginine, ADMA),N(G) -monomethyl-l-arginine (l-NMMA) and N(G) ,N(G') -dimethyl-l-arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l-NMMA inhibit nitric oxide synthase by competing with l-arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high-performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l-NMMA and SDMA. Our assay requires 12 µL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed-mode cation-exchange columns, using synthetic monoethyl-l-arginine as an internal standard. Metabolites were derivatized with ortho-phthaldialdeyhde and 3-mercaptopropionic acid, separated by reverse-phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter-day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l-NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples.

Antioxidant and anti-inflammatory activities of Lonicera japonica Thunb. var. sempervillosa Hayata flower bud extracts prepared by water, ethanol and supercritical fluid extraction techniques.

Lonicera japonica Thunberg (LJ) has long been used as an antipyretic, anti-inflammatory and anti-infectious agent in East Asia. The subspecies L. japonica Thunb. var. sempervillosa Hayata (LJv) is a variant that mainly grows in Taiwan. This study examined the antioxidant and anti-inflammatory activities of the extracts from the flower buds of these two species. The extracts were obtained by three extraction methods: water extraction, ethanol extraction, and supercritical-CO2 fluid extraction (SFE). The antioxidant activities of dry LJ (dLJ) extracts were superior to those of LJv extracts. Water extracts possessed higher activities than that prepared by ethanol or SFE. The total polyphenols content, total flavonoids content, and the amount of chlorogenic acid and luteolin-7-O-glucoside were all higher in the water extracts compared to the other two. The SFE extracts of these two species all exhibited excellent anti-inflammatory activities. Although the water and ethanol extracts of dLJ extracts had higher anti-inflammatory activity than that of LJv extracts, the SFE extracts prepared from fresh LJv flower buds (fLJv) exhibited the highest activity among all extracts. The SFE effectively isolates the bioactive components of L. japonica and can obtain the L. japonica extracts with high anti-inflammatory activity.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes.

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes.

Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma.

BACKGROUND & AIMS: Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease. METHODS: The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages. RESULTS: An accumulation of MDSC was found in mice with HCC irrespective of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL6, IL1ß) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF overexpression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib. CONCLUSIONS: Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.

Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a unique derivative of guanosine 3',5'-cyclic monophosphate (cGMP) formed in mammalian and plant cells in response to production of nitric oxide and reactive oxygen species. 8-Nitro-cGMP possesses signaling activity inherited from parental cGMP, including induction of vasorelaxation through activation of cGMP-dependent protein kinase. On the other hand, 8-nitro-cGMP mediates cellular signaling that is not observed for native cGMP, e.g., it behaves as an electrophile and reacts with protein sulfhydryls, which results in cGMP adduction to protein sulfhydryls (protein S-guanylation). Several proteins have been identified as targets for endogenous protein S-guanylation, including Kelch-like ECH-associated protein 1 (Keap1), H-Ras, and mitochondrial heat shock proteins. 8-Nitro-cGMP signaling via protein S-guanylation of those proteins may have evolved to convey adaptive cellular stress responses. 8-Nitro-cGMP may not undergo conventional cGMP metabolism because of its resistance to phosphodiesterases. Hydrogen sulfide has recently been identified as a potent regulator for metabolisms of electrophiles including 8-nitro-cGMP, through sulfhydration of electrophiles, e.g., leading to the formation of 8-SH-cGMP. Better understanding of the molecular basis for the formation, signaling functions, and metabolisms of 8-nitro-cGMP would be useful for the development of new diagnostic approaches and treatment of diseases related to oxidative stress and redox metabolisms.

Elevated iNOS and 3'-nitrotyrosine in Kaposi's Sarcoma tumors and mouse model.

Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.