bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide and cardiac parasympathetic control in human heart failure
Authors: CHOWDHARY Saqib , NG G. Andre , NUTTALL Sarah L. , COOTE John H. , ROSS Hamish F. , TOWNEND Jonathan N.
Published: August 2015

Cardiac parasympathetic control has prognostic significance in heart failure, but the control mechanisms of this system remain poorly defined. We have demonstrated previously a facilitatory role for nitric oxide (NO) in the parasympathetic control of heart rate in young healthy human subjects. In view of the complex abnormalities of regional NO activity observed in chronic heart failure, we now aim to establish if this mechanism is active in subjects with this condition. Groups of 12heart failure patients [NYHA class II-III; mean age 52 years (range 38-67 years)] and 12 age/sex-matched healthy control subjects [mean age 50 years (range 36-62 years)] were studied. Heart rate variability and baroreflex sensitivity were measured during inhibition of endogenous NO production with NG-monomethyl-l-arginine (l-NMMA; 3mgċh-1ċkg-1) and during administration of an equipressor dose of the control vasoconstrictor phenylephrine (12-36μgċh-1ċkg-1). Basal levels of nitrate+nitrite were measured in the plasma as an indication of systemic NO production. In the heart failure patients, despite an equal rise in blood pressure with both drugs, high-frequency indices of heart rate variability increased less with l-NMMA than with phenylephrine: RMSSD (root mean square of successive RR-interval differences) increased by 4±2 compared with 26±8ms (P < 0.001) and high-frequency power increased by 97±62 compared with 1372±861ms2 (P < 0.001). The increases in cross-spectral baroreflex sensitivity were also lower with l-NMMA than with phenylephrine [high-frequency α-index, 2.2±1.3 and 12.6±3.8ms/mmHg respectively (P < 0.001); low-frequency α-index, 1.3±0.9 and 4.3±1.7ms/mmHg respectively (P < 0.05)]. Healthy subjects showed a similar discrepancy in the response of high-frequency indices of heart rate variability to the two drugs, although baroreflex sensitivity responses were significantly different only for the high-frequency α-index. Levels of plasma nitrate+nitrite were significantly higher in the heart failure patients compared with controls. These data demonstrate that baroreflex-mediated cardiac parasympathetic activation in human heart failure, as in health, is dependent upon endogenous NO synthesis.
Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity
Authors: GROVER Phulwinder K. , RYALL Rosemary L.
Published: August 2015

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65nmol/l using Coulter Counter and [14C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [14C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the γ-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.
A European multicentre, placebo-controlled supplementation study with α-tocopherol, carotene-rich palm oil, lutein or lycopene: analysis of serum responses
Authors: OLMEDILLA Begoña , GRANADO Fernando , SOUTHON Susan , WRIGHT Anthony J.A. , BLANCO Inmaculada , GIL-MARTINEZ Enrique , VAN DEN BERG Henk , THURNHAM David , CORRIDAN Bernice , CHOPRA Mridula , HININGER Isabelle
Published: August 2015

Increased levels of oxidative stress have been implicated in tissue damage and the development of chronic diseases, and dietary antioxidants may reduce the risk of oxidative tissue damage. As part of a European multicentre project, several studies were undertaken with the aim of testing whether the consumption of foods rich in carotenoids reduces oxidative damage to human tissue components. We describe here the serum response of carotenoids and tocopherols upon supplementation with carotenoids from natural extracts (α-carotene+β-carotene, lutein or lycopene; 15mg/day) and/or with α-tocopherol (100mg/day) in a multicentre, placebo-controlled intervention study in 400healthy male and female volunteers, aged 25-45 years, from five European regions (France, Northern Ireland, Republic of Ireland, The Netherlands and Spain). Supplementation with α-tocopherol increased serum α-tocopherol levels, while producing a marked decrease in serum γ-tocopherol. Supplementation with α- + β-carotene (carotene-rich palm oil) resulted in 14-fold and 5-fold increases respectively in serum levels of these carotenoids. Supplementation with lutein (from marigold extracts) elevated serum lutein (approx. 5-fold), zeaxanthin (approx. doubled) and ketocarotenoids (although these were not present in the supplement), whereas lycopene supplementation (from tomato paste) resulted in a 2-fold increase in serum lycopene. The isomer distributions of β-carotene and lycopene in serum remained constant regardless of the isomer composition in the capsules. In Spanish volunteers, additional data showed that the serum response to carotenoid supplementation reached a plateau after 4 weeks, and no significant side effects (except carotenodermia) or changes in biochemical or haematological indices were observed throughout the study. This part of the study describes dose-time responses, isomer distribution, subject variability and side effects during supplementation with the major dietary carotenoids in healthy subjects.
Advanced analysis of spontaneous baroreflex sensitivity, blood pressure and heart rate variability in patients with dilated cardiomyopathy
Authors: MALBERG Hagen , WESSEL Niels , HASART Annett , OSTERZIEL Karl-Josef , VOSS Andreas
Published: August 2015

Baroreflex sensitivity (BRS) is an important parameter in the classification of patients with reduced left ventricular function. This study aimed at investigating BRS in patients with dilated cardiomyopathy (DCM) and in healthy subjects (controls), as well as comparing the values of BRS parameters with parameters of heart rate variability (HRV) and blood pressure variability (BPV). ECG, continuous blood pressure and respiration curves were recorded for 30min in 27 DCM patients and 27 control subjects. The Dual Sequence Method (DSM) includes the analysis of spontaneous fluctuations in systolic blood pressure and the corresponding beat-to-beat intervals of heart rate to estimate bradycardic, opposite tachycardic and delayed baroreflex fluctuations. The number of systolic blood pressure/beat-to-beat interval fluctuations in DCM patients was reduced in comparison with controls (DCM patients: male, 154.4±93.9ms/mmHg; female, 93.7±40.5ms/mmHg; controls: male, 245.5±112.9ms/mmHg; female, 150.6±55.8ms/mmHg, P < 0.05). The average slope in DCM patients was lower than in controls (DCM, 5.3±1.9ms/mmHg; controls, 8.0±5.4ms/mmHg; P < 0.05). Discriminant function analysis showed that, in the synchronous range of the standard sequence method, the DCM and control groups could be discriminated to only 76% accuracy, whereas the DSM gave an improved accuracy of 84%. The combination of six parameters of HRV, BPV and DSM gives an accuracy of classification of 96%, whereas six parameters of HRV and BPV could separate the two groups to only 88% accuracy. Thus the DSM leads to an improved characterization of autonomous regulation in order to differentiate between DCM patients and healthy subjects. BRS in DCM patients is significantly reduced and apparently less effective.
Acute haemodynamic effects of lipolysis-induced increase of free fatty acids in healthy men
Authors: KEARNEY Mark T. , CHOWIENCZYK Philip J. , BRETT Sally E. , SUTCLIFFE Angela , RITTER James M. , SHAH Ajay M.
Published: August 2015

Circulating free fatty acids (FFA) are elevated in subjects with insulin resistance and Type II diabetes, and increase during myocardial ischaemia, but their haemodynamic effects are incompletely understood. During an investigation of the effects of FFA on endothelial function, we administered lipid emulsion (150mgċmin-1 of soybean oil) with heparin (0.2 unitċkg-1ċmin-1) intravenously to eight healthy men for 2h. This increased circulating FFA to 3.1±0.5mmol/l. Forearm blood flow was measured by venous occlusion plethysmography during brachial artery infusions of saline, acetylcholine and nitroprusside before, and at 1 and 2h. Lipid/heparin infusion had no significant effect on vasodilation to nitroprusside but progressively increased responses to acetylcholine (from 6.3±2.0 during 30μgċmin-1 before-lipid infusion to 7.9±1.3 at 1h and 12.2±1.1mlċmin-1ċ100ml-1 at 2h, P < 0.001). Basal flow increased from 2.7±0.7 to 4.7±0.8mlċmin-1ċ100ml-1 from 0 to 2h. We performed a second study to clarify this effect on basal blood flow. Healthy men (n = 8) received, on separate occasions, 4h intravenous infusions of lipid emulsion with heparin and, as a control, saline with heparin. Lipid with heparin increased mean arterial blood pressure (maximum increment 8.2±2.7mmHg, P < 0.01 compared with saline/heparin control) and forearm blood flow (from 1.7±0.2 to 2.9±0.3mlċmin-1ċ100ml-1, P < 0.01) without a significant effect on heart rate, and reduced calculated forearm vascular resistance (from 49.1±5.4 to 31.3±3.9 arbitrary units, P < 0.01). In conclusion, acute elevation of FFA in healthy men increases arterial blood pressure and reduces vascular resistance. These haemodynamic changes could be clinically relevant.
A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria
Authors: YASUI Yumiko , MURANAKA Shikibu , TAHARA Tsuyoshi , SHIMIZU Ryo , WATANABE Sonoko , HORIE Yutaka , NANBA Eiji , UEZATO Hiroshi , TAKAMIYAGI Atsushi , TAKETANI Shigeru , AKAGI Reiko
Published: August 2015

We investigated the molecular defect of the ferrochelatase gene in a Japanese patient with erythropoietic protoporphyria (EPP), and identified a novel 16 base pair (574-589) deletion within exon 5. This deletion resulted in a frame-shift mutation and created a premature stop codon at amino acid position 198. The same molecular defect was also identified in his mother and a brother who had symptomatic EPP, but not in his father who was asymptomatic. The subjects with EPP were homozygous for the low expression haplotype, while his father was heterozygous for this haplotype. These results indicate that the combination of a 16 base pair deletion and low expression of the wild-type allelic variant is responsible for EPP in this pedigree.
Left ventricular long-axis changes in early diastole and systole: impact of systolic function on diastole
Authors: YIP Gabriel W. , ZHANG Yan , TAN Peggy Y. , WANG Mei , HO Pik-Yuk , BRODIN L.-Å. , SANDERSON John E.
Published: August 2015

Impaired long-axis motion is a sensitive marker of systolic myocardial dysfunction, but no data are available that relate long-axis changes in systole with those in diastole, particularly in subjects with diastolic dysfunction and a ‘normal’ left ventricular (LV) ejection fraction. A total of 311 subjects (including 105 normal healthy volunteers) aged 20-89 years with variable degrees of systolic function (LV ejection fraction range 0.15-0.84) and diastolic function were studied using tissue Doppler echocardiography and M-mode echocardiography to determine mean mitral annular amplitude and peak velocity in systole and early and late diastole. The LV systolic mitral annular amplitude (SLAX, where LAX is long-axis amplitude) and peak velocity (Sm) correlated well with the respective early diastolic components (ELAX and Em) and late diastolic (atrial) components (ALAX and Am). A non-linear equation fitted better than a linear relationship (non-linear model: SLAX against ELAX, r2 = 0.67; Sm against Em, r2 = 0.60; SLAX against ALAX and Sm against Am, r2 = 0.42). After adjusting for age, sex and heart rate, linear relationships of early diastolic (ELAX, r2 = 0.70; Em, r2 = 0.60) and late diastolic (ALAX, r2 = 0.61; Am, r2 = 0.64) long-axis amplitudes and velocities with the respective values for SLAX and Sm were found, even in those subjects with apparently ‘isolated’ diastolic dysfunction. Long-axis changes in systole or diastole did not correlate with Doppler mitral velocities. We conclude that ventricular long-axis changes in early diastole are closely related to systolic function, even in subjects with diastolic dysfunction. ‘Pure’ or isolated diastolic dysfunction is uncommon.
Low-protein diet impairs vascular relaxation in virgin and pregnant rats
Authors: KOUMENTAKI Angeliki , ANTHONY Frederick , POSTON Lucilla , WHEELER Timothy
Published: August 2015

Pregnancy is associated with increases in maternal cardiac output and plasma volume and a reduction in peripheral vascular resistance. Cardiac output and plasma volume are substantially reduced in pregnant rats fed a low-protein diet, but it is not known whether vascular function is also compromised. We have investigated vascular function in virgin and pregnant Wistar rats subjected to dietary protein restriction [9% (w/v) casein, compared with 18% (w/v) casein for controls]. The diets were fed to the groups for 18 days; in the pregnant rats, the diets were given from day 1 of pregnancy. Branches of the mesenteric arteries were studied on day 18 of the dietary period using myography. Significant reductions in sensitivity to acetylcholine occurred in vessels from virgin (P = 0.04) and pregnant (P = 0.01) rats that had consumed the 9% casein diet. In arteries from the virgin rats on the restricted diet there was also a significant reduction in sensitivity (P = 0.0003) and maximum relaxation (P = 0.009) to the NO donor spermine NONOate. Mean placental and fetal weights were significantly lower in the rats fed on 9% casein (P<0.0001 and P = 0.005 respectively). Thus low-protein diets impair vasodilator responses in female rats. These effects may contribute to the poor cardiovascular adaptation to pregnancy and lower fetal weights associated with restricted protein intake.
Asynchronous impairment of calcium homoeostasis in different acinar cells after pancreatic duct obstruction in rat
Authors: URUÑUELA Aránzazu , MANSO Manuel A. , DE LA MANO Ana Ma , SEVILLANO Sara , ORFAO Alberto , DE DIOS Isabel
Published: August 2015

Current evidence suggests that alterations within acinar cells are responsible for the development of acute pancreatitis. After inducing acute pancreatitis in rats by pancreatic duct obstruction, we analysed, using flow cytometry, the progressive changes in cytosolic Ca2+ concentrations in individual acinar cells from the earliest stages to 48h after obstruction to investigate whether parallel alterations in the homoeostasis of Ca2+ could be defined in the different acinar cells throughout the evolution of pancreatitis. Morphological alterations of the pancreas, related to the severity of the disease at different stages, were observed by electron microscopy. Hyperamylasaemia and progressively more severe alterations, such as vacuolization, dilatation of endoplasmic reticulum, accumulation of zymogen granules and reorientation towards basolateral membrane, were observed during the first 12h after pancreatic obstruction. A significant increase in cytosolic Ca2+ concentration was measured at these stages in a particular type of acinar cells (R1) differentiated by flow cytometry with low forward scatter (FSC), whereas another representative group of cells (R2) with higher FSC values were able to maintain resting cytosolic Ca2+ concentrations up to 24h after obstruction. Longer periods of pancreatic duct obstruction induced disturbances in Ca2+ homoeostasis in all acinar cells. A similar increase in cytosolic Ca2+ load was reached in both R1 and R2 cells when acute pancreatitis was completely developed. In conclusion, the homoeostasis of Ca2+ in acinar cells is asynchronously impaired during the development of acute pancreatitis; cells with higher FSC (R2) appear to be more resistant than R1 cells.
Increased cationic amino acid flux through a newly expressed transporter in cells overproducing nitric oxide from patients with septic shock
Authors: READE Michael C. , CLARK Megan F. , YOUNG J. Duncan , BOYD C.A.R.
Published: August 2015

Increased production of nitric oxide (NO) is thought to be a factor in the pathogenesis of many human diseases - among them the hypotension that often accompanies sepsis. The supply of the cationic amino acid arginine is known to be rate-limiting for NO production. We hypothesized that cationic amino acid transport might be increased in cells producing excess NO from patients with septic shock. Peripheral blood mononuclear cells were isolated from patients with sepsis and from healthy control subjects. The rates of both NO production and cationic amino acid uptake were increased in cells from patients with septic shock. The increased transport was due almost entirely to an increase in the activity of one transporter, subtype y+. The activity of the other major cationic amino acid transporter (y+L) was unchanged. The expression of CAT2 mRNA, which encodes a y+ transporter protein, was also increased in these cells. We suggest that CAT2 might be a therapeutic target to prevent excess NO production in sepsis and possibly other human disease states, while leaving basal production unchanged.
Molecular forms of adrenomedullin in pericardial fluid and plasma in patients with ischaemic heart disease
Authors: NISHIKIMI Toshio , SHIBASAKI Ikuko , IIDA Hiroshi , ASAKAWA Hiroshi , MATSUSHITA Yasushi , MORI Hideaki , MOCHIZUKI Yoshihiko , OKAMURA Yoshitaka , HORINAKA Shigeo , KANGAWA Kenji , SHIMADA Koichiro , MATSUOKA Hiroaki
Published: August 2015

Experimental studies have demonstrated that adrenomedullin (AM) has a positive inotropic action and exerts inhibitory effects against ventricular remodelling as an autocrine and paracrine factor. However, there is no clinical evidence for AM acting as a local regulator in the human heart. We measured the levels of various molecular forms of AM, i.e. an active form of mature AM (AM-m), an intermediate inactive form of glycine-extended AM (AM-Gly) and total AM (AM-T = AM-m+AM-Gly), in plasma and pericardial fluid using our newly developed immunoradiometric assay in consecutive 67 patients undergoing coronary artery bypass graft surgery. Pericardial fluid and plasma cAMP, atrial natriuretic peptide and brain natriuretic peptide levels were also measured. The relationships between pericardial fluid AM levels and ventricular functions and other hormone levels were analysed. The level of each molecular form of AM in pericardial fluid was closely correlated with that of the other molecular forms of AM in the fluid. However, levels were not correlated with those in plasma. AM-T levels were slightly higher in pericardial fluid than in plasma (+72%; P<0.05), whereas AM-m levels and AM-m/AM-T ratios were markedly higher in pericardial fluid than in plasma (AM-m, +994%; AM-m/AM-T ratio, +443%; both P<0.01). AM-m, AM-Gly and AM-T levels in pericardial fluid were correlated with indices of left ventricular function, and with atrial natriuretic peptide and brain natriuretic peptide levels. Interestingly, AM and cAMP levels were positively correlated in plasma, but negatively correlated in pericardial fluid. In addition, AM-m, AM-Gly and AM-T levels in pericardial fluid were higher in patients with acute coronary syndrome than in those with stable ischaemic heart disease (AM-m, +80%; AM-Gly, +96%; AM-T, +83%; all P<0.01). These results suggest that AM in pericardial fluid reflects cardiac synthesis, and that enhanced cardiac secretion of AM is associated with left ventricular dysfunction, ventricular overload and myocardial ischaemia. Considering that AM has positive inotropic, coronary vasodilatory and anti-remodelling actions, increased cardiac AM may play a compensatory role in the ischaemic and failing myocardium.
Increased nitric oxide elimination from the airways after smoking cessation
Authors: HÖGMAN M. , HOLMKVIST T. , WÅLINDER R. , MERILÄINEN P. , LÚDVÍKSDÓTTIR D. , HÅKANSSON L. , HEDENSTRÖM H.
Published: August 2015

Smokers have been found to have low exhaled nitric oxide (NO) levels. The aim of the present study was to investigate where in the respiratory system the decrease in NO occurs, and whether this decrease was affected by smoking cessation. Measurements of exhaled NO were carried out in smokers (n = 20) and non-smoking control subjects (n = 30). In nine of the smokers, exhaled NO was analysed 1, 2 and 4 weeks after smoking cessation. The level of exhaled NO at a flow rate of 0.1litre/s was significantly lower in smokers (4±2p.p.b.) than in non-smokers (7±5p.p.b.; P = 0.007). A calculation of the contributions from different areas of the lung showed that the NO flux from the airways was significantly lower (14±10 compared with 36±26nl/min; P = 0.0001) and the alveolar fraction was significantly higher (2.1±0.8 compared with 1.5±0.9p.p.b.; P = 0.006) in smokers than in non-smokers. Nine smoking subjects refrained from smoking for 4 weeks, and this resulted in increased NO flux from the airways of 28±17nl/min, which was no longer significantly different from controls. In conclusion, endogenous production of NO in the airways is decreased in smokers, but can be restored to normal values by 4 weeks after cessation of smoking. Smokers have an increased alveolar fraction of NO, and this might be a diagnostic sign of lung damage. Thus NO monitoring can be used to indicate improvements when a smoker decides to stop smoking.
Altered l-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells
Authors: THURAISINGHAM Raj C. , ROBERTS Norman B. , WILKES Mark , NEW David I. , MENDES-RIBEIRO A. Claudio , DODD Susan M. , YAQOOB Muhammad M.
Published: August 2015

Results regarding the nitric oxide (NO) system in uraemia are contradictory. l-Arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. l-Arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.
Enhanced blood insulin overcomes pyruvate dehydrogenase derangements that reflect systemic insulin resistance in obese adolescents
Authors: MOSTERT M. , CERUTTI F. , PICCININI M. , RABBONE I. , SACCHETTI C. , PACINI G. , RINAUDO M.T.
Published: August 2015

Pyruvate dehydrogenase (PDH) has low activity in the circulating lymphocytes (CL) of obese adolescents and adults. In vitro, it is unresponsive to insulin at 5μ-units/ml and is activated at 50μ-units/ml, in contrast with activation and inhibition respectively at these concentrations in CL from controls. These changes are seen as being indicative of a molecular disorder underlying insulin resistance. The aims of the present study were to determine whether a substantial enhancement of blood insulin levels restores the PDH activity in CL from obese adolescents and abolishes the in vitro alterations, and whether PDH activity and indices of insulin resistance are correlated. Six obese adolescents and six normal-weight controls underwent a 4h frequently sampled intravenous glucose test with minimal model analysis, to bring about a sharp rise in blood insulin and provide a reliable index of insulin sensitivity (SI). PDH activity was evaluated in CL obtained from blood samples at set times before and after their exposure to insulin in vitro. Insulin levels rose in all subjects in the first 10min, although to a much greater extent in the obese group, and then decreased until the end of the test (240min; t240). PDH activity in CL paralleled the insulin pattern in the control subjects, whereas in the obese subjects it was below normal 3min before the start of the test (t-3), but rose significantly throughout the test. PDH responses in vitro to insulin in CL taken from the control subjects at t-3 and t240 and in CL taken from the obese subjects at t-3 were as reported above, but were normal (i.e. the same as in control CL) in CL taken from the obese subjects at t240. Baseline PDH activity was inversely correlated with body mass index and with fasting insulin, and directly correlated with SI. These results show that a brief and sharp enhancement of blood insulin overcomes derangements in PDH that reflect systemic insulin resistance in obese adolescents.
Cardiac vagal response to water ingestion in normal human subjects
Authors: ROUTLEDGE Helen C. , CHOWDHARY Saqib , COOTE John H. , TOWNEND Jonathan N.
Published: August 2015

In healthy young subjects there is direct evidence for sympathetic vasoconstrictor activation after drinking water, but this is not accompanied by an increase in arterial blood pressure. A marked pressor response to water ingestion has, however, been observed in elderly subjects and in patients with autonomic failure. We examined the effect of water ingestion on haemodynamic variables and heart rate variability (HRV) markers of cardiac vagal control in ten healthy young subjects and four cardiac transplant recipients with confirmed persistent cardiac vagal denervation. In a random order crossover protocol, changes in heart rate, blood pressure and measures of high frequency (HF) HRV were compared over time following the ingestion of 500ml and 20ml (control) of tap water. In healthy subjects, after drinking 500ml of water the heart rate fell from 67.6±2.0 (mean±S.E.M.) to 60.7±2.4 beats/min (P<0.01), and the bradycardic response peaked between 20 and 25min. There were no significant changes in arterial blood pressure. Over the same time course, water ingestion caused increases in measurements of HF HRV: root-mean-square of successive RR interval differences (RMSSD) increased by 13±2.7ms after 500ml versus 2±3.1ms after 20ml (P<0.05); HF power increased by 686±400 versus -63±322 (P<0.01). In transplant recipients water ingestion was followed by a pressor response (range 13 to 29mmHg). These results provide evidence that water ingestion in normal subjects is followed by an increase in cardiac vagal control that may counteract the pressor effects of sympathetic activation. We suggest that in the elderly, in transplant recipients and in autonomic failure, loss of this buffering mechanism explains the pressor response to drinking water.
Effect of lowering tumour necrosis factor-α on vascular endothelial function in Type II diabetes
Authors: BILSBOROUGH William , O'DRISCOLL Gerard , STANTON Kim , WEERASOORIYA Rukshen , DEMBO Lawrence , TAYLOR Roger , GREEN Daniel
Published: August 2015

Tumour necrosis factor-α (TNFα) is a mediator of reactive oxygen species, which are implicated in endothelial dysfunction and atherosclerosis. Type II diabetes is associated with endothelial dysfunction and elevated circulating TNFα. We hypothesized that reducing serum levels of TNFα, using pentoxifylline, would improve endothelial function. Thirteen subjects [age 58±2 (S.E.M.) years] with Type II diabetes (disease duration 74±13months) undertook a randomized, crossover study of 8weeks pentoxifylline and 8weeks placebo. Endothelium-dependent and-independent vasodilation in resistance arteries was assessed via bilateral forearm venous occlusion plethysmography during intra-brachial infusions of acetylcholine (ACh), sodium nitroprusside (SNP) and NG-monomethyl-l-arginine (l-NMMA). High-resolution ultrasound of the brachial artery in response to ischaemia was used to determine endothelium-dependent conduit vessel flow-mediated dilation (FMD), and endothelium-independent conduit function was assessed by sublingual administration of glyceryl trinitrate (GTN). Serum concentrations of TNFα were also determined. Pentoxifylline lowered serum TNFα from 4.1±0.7 to 2.9±0.6 pg˙ml-1 (P = 0.001). Forearm blood flow (FBF) responses at each dose of ACh did not differ with treatment (P = 0.4). Similarly, FBF responses to SNP (P = 0.8) and l-NMMA (P = 0.2) did not differ. There was also no significant difference in brachial artery diameter during FMD (P = 0.2) or GTN administration (P = 0.06). Despite lowering serum TNFα concentration, pentoxifylline at a dose of 400mg three times a day for 8weeks did not improve vascular function in either conduit or resistance vessels in this group of Type II diabetic subjects.
Erythropoietin and the brain: from neurodevelopment to neuroprotection
Authors: BUEMI M. , CAVALLARO E. , FLOCCARI F. , STURIALE A. , ALOISI C. , TRIMARCHI M. , GRASSO G. , CORICA F. , FRISINA N.
Published: August 2015

It is now widely known that erythropoietin (Epo) does not only affect the haematopoietic system, but it can be considered a multifunctional trophic factor with an effect on the general homoeostasis of the entire organism. The recent discovery of a specific Epo/Epo-receptor system in the central nervous system (CNS) and cerebrospinal fluid, independently of the haematopoietic system, has further paved the way for new studies aimed at investigating the different sites of cerebral expression of Epo and its receptor, the regulation of their expression and, finally, the effects that this hormone has on the development and maturation of the brain. A further aim has been to investigate how it influences CNS homoeostasis and neurotransmission in adult brain. Attention has also been focused on the neurotrophic and neuroprotective function of Epo in different conditions of neuronal damage, such as hypoxia, cerebral ischaemia and subarachnoid haemorrhage, and therefore on the possibility that human recombinant Epo therapy could soon be used in clinical practice, also to limit neuronal damage induced by these diseases.
Dissociation between respiratory effort and dyspnoea in a subset of patients with stroke
Authors: LANINI Barbara , GIGLIOTTI Francesco , COLI Claudia , BIANCHI Roberto , PIZZI Assunta , ROMAGNOLI Isabella , GRAZZINI Michela , STENDARDI Loredana , SCANO Giorgio
Published: August 2015

Dyspnoea is not a prominent complaint of resting patients with recent hemispheric stroke (RHS). We hypothesized that, in patients with RHS presenting abnormalities in respiratory mechanics, increased respiratory motor output could translate into an increased perception of dyspnoea. We studied eight wheelchair-bound patients with RHS (mean age 62.4 years), previously evaluated by computerized tomography scanning, and a control group of normal subjects, matched for age and sex. We assessed routine spirometry, inspiratory and expiratory muscle pressures, breathing pattern and dyspnoea using a modified Borg scale. In six patients, we also measured oesophageal pressure during the maximal sniff manoeuvre and tidal inspiratory swing, and mechanical characteristics of the lung in terms of dynamic elastance during both quiet breathing and a hypercapnic/hyperoxic rebreathing test. During room air breathing, ventilation and tidal volume were similar in patients and controls, while tidal inspiratory swings of oesophageal pressure, an index of inspiratory motor output, were greater in patients (P = 0.005). Patients also exhibited a greater dynamic elastance (P = 0.013). During rebreathing, dynamic elastance remained higher (P = 0.01) and a greater than normal inspiratory motor output was found (P = 0.03). Responses of ventilation and tidal volume to carbon dioxide tension were normal, and in all patients but one a lower Borg score for the unit change in carbon dioxide tension and ventilation was found. In conclusion, a higher than normal inspiratory motor output was unexpectedly associated with a blunted perception of dyspnoea in this subset of RHS patients. This is likely to be due to the modulation of the integration process of respiratory sensation.
18F-Endothelin-1, a positron emission tomography (PET) radioligand for the endothelin receptor system: radiosynthesis and in vivo imaging using microPET
Authors: JOHNSTRÖM Peter , HARRIS Neil G. , FRYER Tim D. , BARRET Olivier , CLARK John C. , PICKARD John D. , DAVENPORT Anthony P.
Published: August 2015

Positron emission tomography (PET) is a powerful technique with the sensitivity to image and quantify receptor-bound radioligands in vivo. Recent progress in PET scanner technology has resulted in the development of dedicated tomographs designed for small animals, with resolution that allows the delineation of discrete organs and their larger substructures in rats and mice. Our aim was to determine whether endothelin-1 (ET-1) could be labelled with 18F, and whether the resulting 18F-ET-1 would have the required pharmacokinetic properties to permit binding and imaging of ET receptors in vivo. 18F-ET-1 could be produced in a total radiochemical yield of 5.9±0.7% in 207±3min (n = 20). Specific radioactivities were in the range 220–370GBq/µmol, and the radiochemical purity of the isolated 18F-ET-1 was >95%. In vivo distribution in the rat was studied using microPET. High levels of 18F-ET-1 uptake were found in lung and kidney, whereas liver showed moderate levels of uptake. The resolution of the microPET scanner was sufficient to differentiate heterogeneous uptake in subrenal structures in the rat.
Three vasoactive peptides, endothelin-1, adrenomedullin and urotensin-II, in human tumour cell lines of different origin: expression and effects on proliferation
Authors: TAKAHASHI Kazuhiro , TOTSUNE Kazuhito , KITAMURO Tomomi , SONE Masahiko , MURAKAMI Osamu , SHIBAHARA Shigeki
Published: August 2015

Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adrenomedullin and urotensin-II, are expressed in various kinds of tumour cells. In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines: T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC-RCW (renal carcinoma). Reverse transcriptase–PCR showed expression of endothelin-1 mRNA in seven out of the eight cell lines, the exception being BeWo cells. ETA receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells. ETB receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and HeLa cells. Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells. Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24h decreased cell numbers to approx. 84% and 90% of control respectively. Treatment with the ETA receptor antagonist BQ-610 (1µM) significantly decreased cell number to about 90% of control, whereas the ETB receptor antagonist BQ-788 had no significant effect. On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1µM) had no significant effects on cell number. These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours. The effect of endothelin-1 on tumour growth appears to be mediated by the ETA receptor.
Pattern of prepro-endothelin-1 expression revealed by reporter-gene activity in kidneys of erythropoietin-overexpressing mice
Authors: SLOWINSKI Torsten , SCHULZ Norma , RUSCHITZKA Frank T. , QUASCHNING Thomas , BAUER Christian , THEURING Franz , NEUMAYER Hans-H. , GASSMANN Max , HOCHER Berthold
Published: August 2015

Transgenic overexpression of erythropoietin (Epo) in mice increases haematocrit to a mean of 80% in adult mice, leading to an increase in blood viscosity and volume. As a consequence, renal tissue endothelin-1 (ET-1) concentrations are significantly elevated in erythropoietin-overexpressing (Epo+) mice (mean±S.E.M; Epo+, 798±71; Epo-, 400±25pg/g tissue; P<0.01). To investigate the pattern of expression of the primary translation product of the ET-1 gene, prepro-ET-1, in kidneys of (Epo+) mice, we generated crossbred mice overexpressing the human EPO gene with mice carrying a reporter gene construct expressing the LacZ gene under the control of the human prepro-ET-1 promotor sequence (LacZ+/Epo+). For comparison, we generated (LacZ+/Epo-) mice from the same strains. After Bluo-Gal staining of frozen kidney sections (n = 10 in each group), intracellular blue precipitates as indicators of prepro-ET-1 promotor activity were detectable in tubular and vascular endothelium and glomerular cells in (LacZ+/Epo-) as well as (LacZ+/Epo+) mice. Comparison of the amount of blue precipitates by semiquantitative scoring showed a significant increase in reporter gene activity in tubular epithelium of (LacZ+/Epo+) mice (mean±S.E.M.; LacZ+/Epo+, 1.64±0.18; LacZ+/Epo-, 1.00±0.19; P<0.05). Reporter gene activity was not significantly elevated in epithelium of small intrarenal arteries of (LacZ+/Epo+) mice (mean±S.E.M.; LacZ+/Epo+, 0.86±0.14; LacZ+/Epo-, 0.38±0.21; P = 0.08) and was similar in glomerular cells (mean±S.E.M.; LacZ+/Epo+, 1.28±0.16; LacZ+/Epo-, 1.14±0.21; P = 0.6). The main source of elevated ET-1 tissue concentration in kidneys of (Epo+) mice therefore seems more likely to be tubular than vascular endothelium or glomerular cells.
Chronic elevated endothelin-1 concentrations regulate mitogen-activated protein kinases ERK 1 and ERK 2 in vascular smooth muscle cells
Authors: BREHM Bernhard R. , KLAUSSNER Martina , WOLF Sabine C.
Published: August 2015

Increased endothelin-1 (ET-1) levels are found in patients with atherosclerosis. ET-1 is known to increase the mitotic response of different growth factors already at threshold concentrations. The aim of this study was to investigate the influence of ET-1 on the mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase (ERK) 1 and ERK 2. Smooth muscle cells were incubated with ET-1 at a concentration of 10-7M for 1–120h. ERK 1 and ERK 2 were determined in cell homogenates by electrophoresis. Specific antibodies were used to investigate the amount of ERK 1 or ERK 2 in the homogenate. The functional activity of ERK 1 and ERK 2 was determined. Immunofluorescence microscopy was performed to analyse the translocation of the MAP kinases into the nucleus. ET-1 incubation for 12h decreased ERK 1 concentration by -51%. After 36h of ET-1 application the concentration of ERK 1 increased to control levels again. When the cells were incubated for 120h ERK 1 rose by +65% above control. The incubation with ET-1 in the presence of an ETA receptor antagonist inhibited the increase of ERK 1. ERK 2 showed a comparable time course with an initial decrease in the protein concentration followed by an increase after 120h. Incubation with an ETA receptor antagonist inhibited the increase in protein concentration after 120h. However, the functional activity of both MAP kinases remained unchanged between 1 and 120h. Especially, after 120h of ET-1 incubation no translocation into the nucleus was observed. However, an additional stimulus with angiotensin II resulted in translocation of ERK into the nucleus. These data show that ET-1 increases the protein concentration of MAP kinases ERK 1 and ERK 2 but not their basal activity. Only an additional stimulation with angiotensin II leads to the translocation of ERK into the cell nucleus.
G-protein-coupled receptors in human atherosclerosis: comparison of vasoconstrictors (endothelin and thromboxane) with recently de-orphanized (urotensin-II, apelin and ghrelin) receptors
Authors: KATUGAMPOLA Sidath D. , KUC Rhoda E. , MAGUIRE Janet J. , DAVENPORT Anthony P.
Published: August 2015

Endothelin (ET)-1 and thromboxane (Tx) levels are increased in human atherosclerosis. One of the aims of this study was to understand how receptors for a peptide mediator (ET-1) with a long physiological half life, would differ from a lipid mediator (TxA2), with a short physiological half life, in human coronary artery disease (CAD). Secondly, to determine if receptor protein is present in human coronary artery vascular smooth muscle for the recently adopted peptide orphan receptors for urotensin-II, apelin and ghrelin. The ETA receptor subtype predominated in the medial smooth muscle layer of both non-diseased coronary artery (NCA) and CAD. However, this subtype was present at relatively low density in the proliferated intimal layer of CAD. The ETB receptor protein was not altered with CAD, compared with NCA. Tx receptor density was significantly (P<0.05) increased in both the media and intima of CAD, compared with NCA. There was no alteration in receptor density, on the medial smooth muscle for urotensin-II and apelin with CAD. Interestingly, receptor density for the novel vasodilator peptide ghrelin was significantly (P<0.05) increased (approx. 4 fold) with CAD, compared with NCA. The alteration of receptor density with disease for Tx and ghrelin provides novel therapeutic targets for the treatment of atherosclerosis. In conclusion, while some GPCR are altered, others remain unchanged with human atherosclerosis. The increase in vasoconstrictor Tx receptor density with disease suggests the importance of Tx receptor antagonism. Intriguingly, the increase in receptor density for the novel vasodilator ghrelin, identified from post-genomic research, may potentially be beneficial with human atherosclerosis.
The ETA receptor antagonist LU 135252 has no electrophysiological or anti-arrhythmic effects during myocardial ischaemia/reperfusion in dogs
Authors: VÁGÓ Hajnalka , SOÓS Pál , ZIMA Endre , GELLÉR László , KÉKESI Violetta , ANDRÁSI Terézia , SZABÓ Tamás , Juhász-Nagy Alexander , MERKELY Béla
Published: August 2015

The anti-arrhythmic effects of ETA receptor antagonists during myocardial ischaemia and reperfusion remain controversial. Moreover, the electrophysiological mechanism has not yet been identified. The aim of this study was to investigate the potential anti-arrhythmic and electrophysiological effects of the ETA receptor antagonist LU 135252 (LU) during myocardial ischaemia and reperfusion in a canine model. A bolus of LU (1mg/kg; n = 10) or saline (control; n = 10) was injected into the left anterior descending coronary artery before ligation of this vessel for 30min, which was followed by a 90-min reperfusion period. LU bolus administration (0.5mg/kg) was repeated every 30min. There were no differences in mean arterial blood pressure or coronary blood flow between the two groups. The determined left ventricular ischaemic mass was 25.5±1.8% and 27.8±2.2% of the total left ventricular mass in the control and LU groups respectively. The total incidence of ventricular fibrillation during ischaemia and reperfusion was 40% in the control and 50% in the LU group (not significantly different). The incidence of non-sustained and sustained ventricular tachycardias during ischaemia, reperfusion and over the whole period (ischaemia plus reperfusion) in the control group was 50%, 50% and 70% respectively, and that in the LU group was 80%, 70% and 100% respectively (no significant differences between groups). The number of ventricular premature beats was not decreased by LU during either ischaemia or reperfusion [median (25th–75th percentile): ischaemia, 20 (13–37) and 56 (32–130) for LU and control groups respectively; reperfusion, 15 (2–21) and 39 (7–74) respectively; ischaemia+reperfusion, 16 (4–35) and 43 (10–82) respectively; no significant differences between groups]. During ischaemia, the monophasic action potential duration at 90% repolarization (MAPD90) decreased significantly, while during reperfusion a significant prolongation of MAPD90 was observed in the left anterior descending region that was similar in the two groups. In conclusion, LU did not affect repolarization changes and did not have anti-arrhythmic effects during either ischaemia or reperfusion in this model.