Effects of sildenafil on nanostructural and nanomechanical changes in mitochondria in an ischaemia‐reperfusion rat model

Sildenafil exerts cardioprotective effects by activating the opening of mitochondrial ATP‐sensitive potassium channels to attenuate ischaemia–reperfusion (IR) injury. In the present study, we used atomic force microscopy (AFM) to investigate changes in mitochondrial morphology and properties to assess sildenafil‐mediated cardioprotection in a rat myocardial infarction model. To investigate the cardioprotective effects of sildenafil, we used an in vivo Sprague‐Dawley rat model of IR. Rats were randomly divided into three groups: (i) sham‐operated rats (control; n = 5); (ii) IR‐injured rats treated with vehicle (normal saline; IR; n = 10); and (iii) IR‐injured rats treated with 0.75 mg/kg, i.p., sildenafil (IR + Sil; n = 10). Morphological and mechanical changes to mitochondria were analysed by AFM. Infarct areas were significantly reduced in sildenafil‐treated rats (7.8 ± 3.9% vs 20.4 ± 7.0% in the sildenafil‐treated and untreated IR groups, respectively; relative reduction 62%; P < 0.001). Analysis of mitochondria by AFM showed that IR injury significantly increased the areas of isolated mitochondria compared with control (24 150 ± 18 289 vs 1495 ± 1139 nm², respectively; P < 0.001), indicative of mitochondrial swelling. Pretreatment with sildenafil before IR injury reduced the mitochondrial areas (7428 ± 3682 nm²; P < 0.001; relative reduction 69.2% compared with the IR group) and ameliorated the adhesion force of mitochondrial surfaces. Together, these results suggest that sildenafil has cardioprotective effects against IR injury in a rat model by improving the morphological and mechanical characteristics of mitochondria.     

Diastolic dysfunction in pulmonary artery hypertension: Creatine kinase and the potential therapeutic benefit of beta‐blockers

Passive properties of the myocardium influence diastolic filling and cardiac output. In heart failure, changes in contributors to the passive properties of the ventricle, such as titin and collagen, and loss of the metabolic enzyme creatine kinase, increase resistance to filling resulting in diastolic dysfunction. Pulmonary artery hypertension (PAH) arises from interactions between the pulmonary vasculature and the right ventricle (RV) which ultimately leads to RV failure. Beta1‐adrenergic receptor blockers (BB) act on the myocardium and are beneficial in left heart failure but are not used in PAH. We investigated whether BB improved survival and RV function in a rat model of PAH. Rats were injected with monocrotaline (60 mg/kg) to induce PAH and RV failure, or saline as controls (CON). When PAH was established, rats were treated with metoprolol (10 mg/kg per day) (MCT+BB) or vehicle (sucrose) (MCT); CON were treated with vehicle. In vivo measurement of RV compliance using pressure–volume catheter, indicated diastolic dysfunction in the RV of MCT rats was improved with BB treatment. Expression of creatine kinase protein and mRNA was lower in MCT rats compared to CON, with a trend for reversion by BB treatment. Isolated CON RV myocytes had a positive contraction response to faster pacing, whereas it was negative in MCT. MCT+BB cells had an intermediate response, indicating improved ability to respond to increased demand. BB improved diastolic function, partially restored metabolic enzymes and augmented contractility in PAH. These data support the hypothesis that BB may be beneficial in PAH by supporting RV function.     

Chemopreventive and anti‐angiogenic effects of dietary phenethyl isothiocyanate in an N‐methyl nitrosourea‐induced breast cancer animal model

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen‐dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N‐methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor‐free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC‐treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC‐treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC‐treated animals. These studies indicate that supplementation with PEITC prolongs the tumor‐free survival, reduces tumor incidence and burden, and is chemoprotective in NMU‐induced estrogen‐dependent breast cancer in rats. For the first time, it is reported that PEITC has anti‐angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Reperfusion Therapy with Low‐Dose Insulin or Insulin‐Like Growth Factor 2; Myocardial Function and Infarct Size in a Porcine Model of Ischaemia and Reperfusion

In an open‐chest porcine model, we examined whether myocardial pharmacological conditioning at the time of reperfusion with low‐dose insulin or insulin‐like growth factor 2 (IGF2), not affecting serum glucose levels, could reduce infarct size and improve functional recovery. Two groups of anaesthetized pigs with either 60 or 40 min. of left anterior descending artery occlusion (total n = 42) were randomized to receive either 0.9% saline, insulin or IGF2 infusion for 15 min., starting 5 min. before a 180‐min. reperfusion period. Repeated fluorescent microsphere injections were used to confirm ischaemia and reperfusion. Area at risk and infarct size was determined with Evans blue and triphenyltetrazolium chloride staining. Local myocardial function was evaluated with multi‐layer radial tissue Doppler strain and speckle‐tracking strain from epicardial echocardiography. Western blotting and TUNEL staining were performed to explore apoptosis. Infarct size did not differ between treatment groups and was 56.7 ± 6.8%, 49.7 ± 9.6%, 56.2 ± 8.0% of area at risk for control, insulin and IGF2 group, respectively, in the 60‐min. occlusion series. Corresponding values were 45.6 ± 6.0%, 48.4 ± 7.2% and 34.1 ± 5.8% after 40‐min. occlusion. Global and local cardiac function did not differ between treatment groups. No differences related to treatment could be found in myocardial tissue cleaved caspase‐3 content or the degree of TUNEL staining. Reperfusion therapy with low‐dose insulin or with IGF2 neither reduced infarct size nor improved function in reperfused myocardium in this in vivo porcine model.     

l‐Carnitine Attenuates Cardiac Remodelling rather than Vascular Remodelling in Deoxycorticosterone Acetate‐Salt Hypertensive Rats

Abstract: l ‐Carnitine is an important co‐factor in fatty acid metabolism by mitochondria. This study has determined whether oral administration of l ‐carnitine prevents remodelling and the development of impaired cardiovascular function in deoxycorticosterone acetate (DOCA)‐salt hypertensive rats (n = 6–12; #p < 0.05 versus DOCA‐salt). Uninephrectomized rats administered DOCA (25 mg every 4th day s.c.) and 1% NaCl in drinking water for 28 days developed cardiovascular remodelling shown as systolic hypertension, left ventricular hypertrophy, increased thoracic aortic and left ventricular wall thickness, increased left ventricular inflammatory cell infiltration together with increased interstitial collagen and increased passive diastolic stiffness and vascular dysfunction with increased plasma malondialdehyde concentrations. Treatment with l ‐carnitine (1.2% in food; 0.9 mg/g/day in DOCA‐salt rats) decreased blood pressure (DOCA‐salt 169 ± 2; + l ‐carnitine 148 ± 6# mmHg), decreased left ventricular wet weights (DOCA‐salt 3.02 ± 0.07; + l ‐carnitine 2.72 ± 0.06# mg/g body‐wt), decreased inflammatory cells in the replacement fibrotic areas, reduced left ventricular interstitial collagen content (DOCA‐salt 14.4 ± 0.2; + l ‐carnitine 8.7 ± 0.5# % area), reduced diastolic stiffness constant (DOCA‐salt 26.9 ± 0.5; + l ‐carnitine 23.8 ± 0.5# dimensionless) and decreased plasma malondialdehyde concentrations (DOCA‐salt 26.9 ± 0.8; + l ‐carnitine 21.2 ± 0.4# μmol/l) without preventing endothelial dysfunction. l ‐carnitine attenuated the cardiac remodelling and improved cardiac function in DOCA‐salt hypertension but produced minimal changes in aortic wall thickness and vascular function. This study suggests that the mitochondrial respiratory chain is a significant source of reactive oxygen species in the heart but less so in the vasculature in DOCA‐salt rats, underlying the relatively selective cardiac responses to l ‐carnitine treatment.     

Protective cardiorenal effects of spironolactone in a rodent model of polycystic kidney disease

Studies were performed to examine the contribution of aldosterone to the pathogenesis of cardiovascular and renal disease in a rodent model of genetic kidney disease. Spironolactone (20 mg/kg per day) was administered in water to mixed sex Lewis Polycystic Kidney (LPK) rats (n = 20) and control Lewis rats (n = 27) from 4 to 12 weeks of age. At 12 weeks of age, hypertension was reduced in female LPK rats; systolic blood pressure declined from 226.4 ± 26.8 mmHg in untreated rats and to 179.2 ± 3.2 mmHg in treated rats (P = 0.018). No similar effect on male or control rats was found. Water consumption and urine volume were significantly greater in LPK animals than in Lewis rats, and treatment reduced both variables by ~30% in LPK animals (P < 0.05). Proteinuria and the urinary protein‐to‐creatinine ratio were normalized in treated LPK relative to Lewis controls, and plasma creatinine levels were significantly reduced by treatment in LPK rats. Spironolactone did not alter kidney morphology in LPK rats (fibrosis or cyst size). Aortic vascular responses to noradrenaline and acetylcholine were sensitized and impaired in the LPK (P < 0.01). Aldosterone antagonism did not alter these responses or indicators of aortic structural remodelling. There was no treatment effect on left ventricular hypertrophy or elevated cardiac messenger RNA for β‐myosin‐heavy chain and brain natriuretic peptide in the LPK rats. However, perivascular fibrosis and messenger RNA for α‐cardiac actin were normalized by spironolactone in LPK animals relative to Lewis controls. In conclusion, we have shown an important blood pressure independent effect whereby inhibition of aldosterone via spironolactone was able to retard both renal and cardiac disease progression in a rodent model of polycystic kidney disease.     

Arrhythmic effects of Epac‐mediated ryanodine receptor activation in Langendorff‐perfused murine hearts are associated with reduced conduction velocity

Recent papers have attributed arrhythmic substrate in murine RyR2‐P2328S hearts to reduced action potential (AP) conduction velocities (CV), reflecting acute functional inhibition and/or reduced expression of sodium channels. We explored for acute effects of direct exchange protein directly activated by cAMP (Epac)‐mediated ryanodine receptor‐2 (RyR2) activation on arrhythmic substrate and CV. Monophasic action potential (MAP) recordings demonstrated that initial steady (8 Hz) extrinsic pacing elicited ventricular tachycardia (VT) in 0 of 18 Langendorff‐perfused wild‐type mouse ventricles before pharmacological intervention. The Epac activator 8‐CPT (8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate) (VT in 1 of 7 hearts), and the RyR2 blocker dantrolene, either alone (0 of 11) or with 8‐CPT (0 of 9) did not then increase VT incidence (P>.05). Both progressively increased pacing rates and programmed extrasystolic (S2) stimuli similarly produced no VT in untreated hearts (n=20 and n=9 respectively). 8‐CPT challenge then increased VT incidences (5 of 7 and 4 of 8 hearts respectively; P<.05). However, dantrolene, whether alone (0 of 10 and 1 of 13) or combined with 8‐CPT (0 of 10 and 0 of 13) did not increase VT incidence relative to those observed in untreated hearts (P>.05). 8‐CPT but not dantrolene, whether alone or combined with 8‐CPT, correspondingly increased AP latencies (1.14±0.04 (n=7), 1.04±0.03 (n=10), 1.09±0.05 (n=8) relative to respective control values). In contrast, AP durations, conditions for 2:1 conduction block and ventricular effective refractory periods remained unchanged throughout. We thus demonstrate for the first time that acute RyR2 activation reversibly induces VT in specific association with reduced CV.     

Ventricular repolarization in a rat model of global heart failure

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Effects of JTV‐519 on stretch‐induced manifestations of mechanoelectric feedback

JTV‐519 is a 1,4‐benzothiazepine derivative with multichannel effects that inhibits Ca²⁺ release from the sarcoplasmic reticulum and stabilizes the closed state of the ryanodine receptor, preventing myocardial damage and the induction of arrhythmias during Ca²⁺ overload. Mechanical stretch increases cellular Na⁺ inflow, activates the reverse mode of the Na⁺/Ca²⁺ exchanger, and modifies Ca²⁺ handling and myocardial electrophysiology, favoring arrhythmogenesis. This study aims to determine whether JTV‐519 modifies the stretch‐induced manifestations of mechanoelectric feedback. The ventricular fibrillation (VF) modifications induced by acute stretch were studied in Langendorff‐perfused rabbit hearts using epicardial multiple electrodes under control conditions (n=9) or during JTV‐519 perfusion: 0.1 μmol/L (n=9) and 1 μmol/L (n=9). Spectral and mapping techniques were used to establish the baseline, stretch and post‐stretch VF characteristics. JTV‐519 slowed baseline VF and decreased activation complexity. These effects were dose‐dependent (baseline VF dominant frequency: control=13.9±2.2 Hz; JTV 0.1 μmol/L=11.1±1.1 Hz, P<.01; JTV 1 μmol/L=6.6±1.1 Hz, P<.0001). The stretch‐induced acceleration of VF (control=38.8%) was significantly reduced by JTV‐519 0.1 μmol/L (19.8%) and abolished by JTV 1 μmol/L (−1.5%). During stretch, the VF activation complexity index was reduced in both JTV‐519 series (control=1.60±0.15; JTV 0.1 μmol/L=1.13±0.3, P<.0001; JTV 1 μmol/L=0.57±0.21, P<.0001), and was independently related to VF dominant frequency (R=.82; P<.0001). The fifth percentile of the VF activation intervals, conduction velocity and wavelength entered the multiple linear regression model using dominant frequency as the dependent variable (R=−.84; P<.0001). In conclusion, JTV‐519 slowed and simplified the baseline VF activation patterns and abolished the stretch‐induced manifestations of mechanoelectric feedback.     

Preparation of 68Ga‐Mg‐Ca‐phytate colloid and its evaluation as a liver imaging agent

The objective of this study was to investigate the radiosynthesis of ⁶⁸Ga‐Mg‐Ca‐phytate colloid and then characterise the formulation for radiochemical purity (RCP), radioactive particle size distribution, and biodistribution in normal rats. This radiocolloid was prepared by mixing an aqueous solution of phytic acid, ⁶⁸Ga³⁺ ions, a dispersant, Mg²⁺ and Ca²⁺ ions, and then heating the contents at 100°C for 5 minutes. After cooling the vial to 5°C, the solution was basified to pH 5 and stored in the cold. The resulting product contained 92±3% RCP ⁶⁸Ga‐colloidal particles and a low level (8±3%) of soluble ⁶⁸Ga‐Mg‐Ca‐phytate. Particle size experiments defined the radioactive particle population was 6±4% <20 nm, 90±6% 20 to 200 nm, and 4% were >200 nm in diameter. Intravenous injection of the ⁶⁸Ga‐colloid dispersion to rats resulted in 93% uptake by the liver plus spleen, 1% lungs, 1% total blood, and 6% in the carcass after 20 minutes. This optimal formulation remained stable at 5°C for 1½ hours in vitro, and it resulted in the same biodistribution as the formulation prepared at t  = 0 hours. The preclinical data so far indicate that ⁶⁸Ga‐Mg‐Ca‐colloid has excellent potential as a liver imaging agent.     

Effects of inorganic nitrate in a rat model of monocrotaline‐induced pulmonary arterial hypertension

The nitrate‐nitrite‐nitric oxide (NO) pathway represents an alternative source of NO generation, which is independent of NO synthase and potentiated by hypoxia. Augmentation of this pathway by dietary nitrate has proven favourable effects in several cardiovascular disease models. However, less is known regarding its potential value in pulmonary arterial hypertension (PAH). The aim of this study was to assess the effects of oral inorganic nitrate administration in monocrotaline (MCT)‐induced PAH. Male 12‐week‐old Wistar rats were injected subcutaneously with monocrotaline (MCT, 60 mg/kg). Nitrate treatment (0.3 or 1 mmol/kg/d; drinking water) commenced on day 12 following the MCT injection and continued for 16 days. Nitrate administration did not attenuate right ventricular (RV) hypertrophy, increased lung weight and up‐regulated mRNA expression of brain natriuretic peptide. Plasma nitrate and nitrite levels were significantly increased as well as lung nitrate level, whereas nitrite lung level was decreased following nitrate treatment (1 mmol/kg/d). MCT‐induced PAH resulted in an increased MnSOD protein level, which was not observed following nitrate treatment. MCT‐associated up‐regulation of nNOS in the lung appeared to be dose‐dependently prevented by nitrate treatment. Western blot analysis did not reveal any differences in eNOS, iNOS, XO or gp91phox expression in the lungs among the groups. In conclusion, nitrate treatment did not significantly attenuate pathological RV and lung remodelling in the rat MCT model of PAH. The suppression of MnSOD and nNOS expression by nitrate could be interpreted as reduced demand of endogenous antioxidant defence in this model.     

Renal cellular hypoxia in adenine‐induced chronic kidney disease

We determined whether adenine‐induced chronic kidney disease (CKD) in rats is associated with renal tissue hypoxia. Adenine (100 mg) or its vehicle was administered to male Sprague‐Dawley rats, daily by oral gavage, over a 15‐day period. Renal function was assessed before, and 7 and 14 days after, adenine treatment commenced, by collection of a 24‐hour urine sample and a blood sample from the tail vein. On day 15, arterial pressure was measured in conscious rats via the tail artery. Renal tissue hypoxia was then assessed by pimonidazole adduct immunohistochemistry and fibrosis was assessed by staining tissue with picrosirius red and Masson's trichrome. CKD was evident within 7 days of commencing adenine treatment, as demonstrated by increased urinary albumin to creatinine ratio (30 ± 12‐fold). By day 14 of adenine treatment plasma creatinine concentration was more than 7‐fold greater, and plasma urea more than 5‐fold greater, than their baseline levels. On day 15, adenine‐treated rats had slightly elevated mean arterial pressure (8 mmHg), anaemia and renomegaly. Kidneys of adenine‐treated rats were characterised by the presence of tubular casts, dilated tubules, expansion of the interstitial space, accumulation of collagen, and tubulointerstitial hypoxia. Pimonidazole staining (hypoxia) co‐localised with fibrosis and was present in both patent and occluded tubules. We conclude that renal tissue hypoxia develops rapidly in adenine‐induced CKD. This model, therefore, should prove useful for examination of the temporal and spatial relationships between tubulointerstitial hypoxia and the development of CKD, and thus the testing of the ‘chronic hypoxia hypothesis’.     

Dose‐dependent effects of sildenafil on post‐ischaemic left ventricular function in the rat isolated heart

Objectives Sildenafil may be beneficial during myocardial ischaemia/reperfusion, but this effect may be dose‐dependent, accounting for previous conflicting results. We have explored the effects of two acute and one chronic administration regimen on left ventricular function. Methods The study was conducted on 36 Wistar rats (290 ± 7 g). Sildenafil was administered 30 min before ischaemia at a low (0.7 mg/kg, n= 8) or high (1.4 mg/kg, n= 8)dosage. The chronic treatment arm (n= 8) consisted of two daily injections of sildenafil (0.7 mg/kg) for three weeks. The control group was formed by 12 rats. Ischaemic contracture, post‐ischaemic recovery and hypercontracture were measured in isolated, Langendorff‐perfused preparations. Key findings Ischaemic contracture tended to be lower after high‐dose sildenafil, while remaining unchanged after low‐dose or chronic sildenafil administration. Compared with controls (62.9 ± 2.0% of baseline developed pressure), post‐ischaemic recovery was higher (P= 0.0069) after low dose (75.1 ± 2.4%), unchanged (P= 0.13) after high dose (69.1 ± 2.1%), but lower (P < 0.001) after chronic (42.9 ± 4.5%) sildenafil administration. Compared with controls (71.8 ± 3.9 mmHg), hypercontracture was higher (P= 0.0052) after chronic sildenafil administration (89.5 ± 4.1 mmHg), but similar after acute low dose (65.7 ± 3.3 mmHg, P= 0.33) or high dose (67.1 ± 4.7 mmHg, P= 0.43). Conclusions The effects of sildenafil after ischaemia/reperfusion were strongly dose‐dependent. Beneficial actions on left ventricular function were evident after acute pretreatment with a low dosage, but were lost after doubling the dose. Chronic sildenafil administration deteriorated left ventricular function during ischaemia and reperfusion.     

Non‐Clinical Pharmacokinetics,Prediction of Human Pharmacokinetics and First‐in‐Human Dose Selection for CNTO 5825, an Anti‐Interleukin‐13 Monoclonal Antibody

CNTO 5825 is a human anti‐interleukin‐13 (IL‐13) monoclonal antibody (mAb) that inhibits binding of human IL‐13 to IL‐13Rα1 and IL‐13Rα2. The purpose of this investigation was to predict human pharmacokinetics (PK) of CNTO 5825 using different allometric approaches and non‐clinical PK data in order to select the right and safe doses for first‐in‐human (FIH) study. After intravenous (IV) administration of CNTO 5825, clearance (CL) ranged from 9.98 to 11.49 ml/day/kg in rats and from 5.78 to 7.19 ml/day/kg in cynomolgus monkeys. The volume of distribution at steady‐state (Vss) in rats was large (151.52–155.64 ml/kg) compared to cynomolgus monkey (49.77–61.10 ml/kg). The terminal half‐life (T_1/2) ranged from 12.29 to 14.15 days in rats and from 6.61 to 7.73 days in cynomolgus monkeys. The PK of CNTO 5825 was linear in 1–10 mg/kg dose range in both species. The bioavailability after subcutaneous (SC) administration was 94% and 79% in rats and cynomolgus monkeys, respectively. The predicted CL and Vss based on allometric methods and PK data from rats and monkeys were within twofold of observed CL and Vss in human beings; the predicted CL and Vss in human beings (70 kg) based on time‐invariant method with combined PK data from rats and monkeys were 4.84 ± 1.13 ml/day/kg and 68.93 ± 35.55 ml/kg, respectively. The selected doses for the FIH study based on time‐invariant method and no observed adverse effect level in toxicity studies in rats and monkeys provided exposures that were subsequently shown to be well tolerated and safe in human beings.     

Remote per‐conditioning reduces oxidative stress,downregulates cyclo‐oxygenase‐2 expression and attenuates ischaemia–reperfusion‐induced acute kidney injury

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Pharmacokinetics of the Experimental Non‐Nucleosidic DNA Methyl Transferase Inhibitor N‐Phthalyl‐l‐Tryptophan (RG 108) in Rats

DNA methyl transferase (DNMT) inhibitors can re‐establish the expression of tumour suppressor genes in malignant diseases, but might also be useful in other diseases. Inhibitors in clinical use are nucleosidic cytotoxic agents that need to be integrated into the DNA of dividing cells. Here, we assessed the in vivo kinetics of a non‐nucleosidic inhibitor that is potentially free of cytotoxic effects and does not require cell division. The non‐specific DNMT inhibitor N‐phthalyl‐l ‐tryptophan (RG 108) was injected subcutaneously in rats. Blood was drawn 0, 0.5, 1, 2, 4, 6, 8 and 24 hr after injection and RG 108 in plasma was measured by high‐performance liquid chromatography coupled to mass spectrometry. Trough levels and area under the curve (AUC) were significantly higher with multiple‐dose administration and cytochrome inhibition. In this group, time to maximal plasma concentration (t_max, mean ± S.D.) was 37.5 ± 15 min., terminal plasma half‐life was approximately 3.7 h (60% CI: 2.1–15.6 h), maximal plasma concentration (C_max) was 61.3 ± 7.6 μM, and AUC was 200 ± 54 μmol·h/l. RG 108 peak levels were not influenced by cytochrome inhibition or multiple‐dose administration regimens. Maximal tissue levels (C_max in μmol/kg) were 6.9 ± 6.7, 1.6 ± 0.4 and 3.4 ± 1.1 in liver, skeletal and heart muscle, respectively. We conclude that despite its high lipophilicity, RG 108 can be used for in vivo experiments, appears safe and yields plasma and tissue levels in the range of the described 50% inhibitory concentration of around 1 to 5 μM. RG 108 can therefore be a useful tool for in vivo DNMT inhibition.     

Physiological mechanisms for the increase in renal solute‐free water clearance by a glucagon‐like peptide‐1 mimetic

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Increased brain l‐arginine availability facilitates cutaneous heat loss induced by running exercise

The effects of increased brain availability of l ‐arginine (l ‐arg), a precursor for nitric oxide synthesis, on core body temperature (T_core) and cutaneous heat loss were evaluated in running rats. One week prior to the experiments, adult male Wistar rats received the following implants: a chronic guide cannula in the lateral cerebral ventricle and a temperature sensor in the abdominal cavity. On the day of the experiments, the rats were assigned to receive a 2‐μL intracerebroventricular injection of either NaCl (0.15 mol/L) or l ‐arg solution (0.825, 1.65 or 3.30 mol/L); T_core and tail skin temperature were measured while the rats ran at a speed of 18 m/min until they were fatigued. l ‐arginine induced a dose‐dependent reduction in the threshold T_core required for cutaneous heat loss (38.09 ± 0.20°C for 3.30‐mol/L l ‐arg vs 38.61 ± 0.10°C for saline; P < 0.05), which attenuated the exercise‐induced hyperthermia. Although the rats treated with l ‐arg presented a lower T_core at the end of exercise (~0.7°C lower after treatment with the highest dose), no changes in the time to fatigue were observed relative to the control trial. These results suggest that brain l ‐arg controls heat loss during exercise, most likely by modulating the sympathetic vasoconstrictor tonus to skin vessels. Furthermore, despite facilitating cutaneous heat loss mechanisms, increased brain l ‐arg availability did not enhance physical performance.     

In Utero Exposure to Diethylstilboestrol or 4‐n‐Nonylphenol in Rats: Number of Sertoli Cells,Diameter and Length of Seminiferous Tubules Estimated by Stereological Methods

Abstract: The effects on testis weight and histopathology were studied in 11‐day‐old male Wistar rats after prenatal exposure to peanut oil (control), diethylstilboestrol 30 μg/kg b.wt./day, or 4‐n‐nonylphenol 75 mg/kg b.wt./day from gestational day 11 to 18. Additionally, the diameter and length of seminiferous tubules, and the number of Sertoli cells were investigated with stereological methods. Such unbiased methods have not previously been applied on testis diameter and length or on Sertoli cell number of 11‐day‐old rats. In the control group, the mean length of the seminiferous tubule was 3.0 m±0.6, the mean diameter of the seminiferous tubule was 83 μm±6, and the mean number of Sertoli cells was 26.1×10⁶±4.6. No differences in testis weight, histopathology, or length or diameter of the seminiferous tubules were observed in the diethylstilboestrol and nonylphenol exposed groups when compared to the control group. In the diethylstilboestrol‐treated group, a statistically significant decrease in the number of Sertoli cells was observed (P<0.01) when compared to the control group, whereas nonylphenol had no effect. The result suggests that diethylstilboestrol decreases Sertoli cell proliferation in the foetal testis and furthermore indicate that oestrogens may pose a risk to the reproductive capacity in sensitive species, including man.     

Elevated plasma Pim‐1 and its clinical significance in patients with pulmonary arterial hypertension

This study was aimed to determine plasma Pim‐1 levels in patients with pulmonary arterial hypertension (PAH) and to estimate the clinical value of Pim‐1 as a biomarker of PAH. This was a single‐centre retrospective study in 111 patients with congenital heart disease (CHD) and idiopathic PAH (IPAH). Those CHD patients were divided into two groups: PAH associated with CHD (PAH‐CHD) and CHD without PAH (nPAH‐CHD). Plasma Pim‐1 levels were measured by enzyme‐linked immunosorbent assay. (a) Plasma Pim‐1 levels were significantly increased in patients with PAH‐CHD and IPAH compared with the healthy control group (27.81 ± 11.34 ng/mL vs 13.02 ± 5.30 ng/mL; 32.81 ± 12.28 ng/mL vs 13.02 ± 5.30 ng/mL, P < 0.05) and nPAH‐CHD (27.81 ± 11.34 ng/mL vs 17.33 ± 7.99 ng/mL; 32.81 ± 12.28 ng/mL vs 17.33 ± 7.99 ng/mL, P < 0.05). Pim‐1 levels were substantially increased in patients with severe PAH‐CHD compared with mild‐to‐moderate PAH‐CHD (19.12 ± 6.70 ng/mL vs 8.54 ± 3.71 ng/mL, P < 0.05). (b) Pim‐1 levels were correlated positively with the mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR) (r = 0.582, 0.516; P < 0.001, respectively), while negatively with tricuspid annular plane systolic excursion (TAPSE), tricuspid annular plane systolic velocity (S’) and right ventricular fractional area changes (RVFAC) (r = ?0.375, ?0.354, ?0.507; P < 0.05, respectively). (c) PAH‐CHD and severe PAH‐CHD was identified by plasma Pim‐1 with a cutoff value of 16.8 ng/mL (P < 0.001) with a sensitivity of 87.3% and a specificity of 65%, and a cutoff value of 20.53 ng/mL (P < 0.001) with a sensitivity of 87.3% and a specificity of 52%, respectively. Plasma Pim‐1 levels were significantly higher in patients with PAH‐CHD and IPAH. Plasma Pim‐1 may represent an effectively biomarker in patients with PAH.