Pioglitazone Alters Ace/Ace 2 Balance to Favour Ace2 Independently Of Glycaemia Levels in Diabetic Rat Heart.

The activation of the renin-angiotensin system (RAS) contributes to the pathogenesis of cardiac damage during diabetes. In the present study, we investigated the role of pioglitazone, dapagliflozin and their combination on RAS components in streptozotocin-induced diabetic cardiomyopathy in Wistar rats. Blood glucose, serum lipids, and ACE (angiotensin-converting enzyme), ACE2 levels were determined. mRNA levels of Myh6 (myosins heavy chain), Myh7, Ace, Ace2, Nppa, Nppb (natriuretic peptide A, B) and Ppars (peroxisome proliferator activating receptors) genes in the heart were determined by real-time PCR (polymerase chain reaction). Protein expression of ACE and ACE2 was assessed by western blotting. After six weeks pioglitazone suppressed Ace mRNA and protein levels (p<0.05) and modified the Ace/Ace2 ratio (p<0.05) in the cardiac tissue of diabetic rats. Pioglitazone significantly decreased serum lipids (p<0.05) but did not significantly influence blood glucose and ACE serum levels of diabetic animals. Dapagliflozin had a significant glucose-lowering action (p<0.05) however, it had no impact on the Ace/Ace2 ratio. The combination of both compounds markedly improved blood glucose (p<0.05) as well as the Myh6/Myh7 ratio (p<0.05) but had no further impact on the Ace to Ace2 balance in cardiac tissue compared to pioglitazone monotherapy. We found that pioglitazone improves the cardiac Ace/ Ace2 ratio in diabetic rats suggesting a potential cardioprotective effect. This effect is independent of its antidiabetic and metabolic effects.

Proapoptotic and antiapoptotic stimuli alternation in myocardial infarction experimental model.

Myocardial infarction is a life-threatening complication of the coronary artery disease - the leading cause of premature death worldwide. The severity of this condition is the result of cellular death following the myocardial ischaemia, which occurs via several mechanism including apoptosis. For the research of this condition, animal models are often employed. We established isoprenaline-induced rat model of myocardial infarction, focusing on the immunohistochemical analysis of the expression of antiapoptotic and proapoptotic proteins BCL-2 and BAX, respectively. Apoptosis (based on BAX-positivity) was activated in cardiac muscle cells within the first day, later on day 8 also in fibroblasts of the forming scar tissue. Antiapoptosis in cardiac muscle cells was weak to moderate on the day 1 and 2, on the day 8 macrophages were strongly positive for BCL-2. The results confirmed that programmed cell death as well as mechanisms of antiapoptosis contribute to the pathogenesis of myocardial infarction. Previous research demonstrated that by experimentally affecting proapoptotic and antiapoptotic signals, it is possible to influence various aspects of myocardial infarction including: infarction size, cardiac remodelling and prognosis of the heart failure. Future research is warranted to fully elucidate the role of this process during myocardial infarction, which will result in refined diagnostic and therapeutic strategies (Tab. 1, Fig. 1, Ref. 21). Keywords: myocardial infarction, isoprenaline, apoptosis, necrosis, BCL-2, BAX.

Aortic butyrylcholinesterase is reduced in spontaneously hypertensive rats.

Despite the fact that vessels have sparse cholinergic innervation, acetylcholine (ACh), the primary neurotransmitter of parasympathetic nervous system, has been commonly used in physiological experiments to assess vascular function. ACh is hydrolyzed by two cholinesterases (ChE), namely acetylcholin-esterase and butyrylcholinesterase (BChE). However, little is known about these enzymes in blood vessels. The aim of the project was to characterize the expression and activity of ChE in rat aorta. As the effect of ACh on vascular tone depends on the presence of endothelium, Wistar rats were used as a model with intact endothelium and spontaneously hypertensive rats as a model of impaired endothelial function. Relative expressions of both ChE in different parts of the aorta were determined using RT-qPCR. Enzyme activities were assessed in tissue homogenates by Ellman's assay. Here we showed that both ChE are present in each part of rat aorta, while mRNA is more abundant for BChE than for AChE, irrespective of aortic compartment or genotype. Normotensive Wistar rats possess higher aortic mRNA expression and activity of BChE compared to SHR. We concluded that BChE is the dominant type of ChE in rat aorta and it might play an important role in the regulation of vascular tone.

Altered serum levels of neprilysin in heart failure patients with reduced ejection fraction.

OBJECTIVE: In addition to the recent success of neprilysin inhibition in treatment of heart failure, elevated soluble neprilysin (sNEP) in circulation has been suggested to be a prognostic biomarker in heart failure with a reduced ejection fraction (HFrEF). However, the diagnostic performance of sNEP is nebulous and its levels in HFrEF have not been compared with controls. For the purpose of this study, we determined the role of sNEP levels as a biomarker in routine ambulatory care of HFrEF patients, when compared to the control subjects. METHODS: Ambulant patients with chronic HFrEF (n = 18) were included. Apparently healthy volunteers - hospital physicians (n = 9) were included as the controls. Besides standard diagnostic tools (echocardiographic examination and laboratory biochemical diagnostic tests including NT-proBNP assessment), we analysed serum levels of neprilysin with a commercially available human soluble neprilysin ultrasensitive ELISA kit (Aviscera Bioscience, USA). RESULTS: Concentrations of sNEP were significantly reduced in HFrEF patients (average ± S.E.M.=1038 ± 464 pg/ml) when compared to the controls (1947 ± 613 pg/ml; p < 0.05). Two of eighteen HFrEF samples were below, while two of ten control samples were above the detection limit of the immunoassay. We documented a lack of significant correlation between sNEP and left ventricular ejection fraction (LVEF) and other echocardiographic features as well as NT-proBNP. However, sNEP significantly negatively correlated to serum natrium levels (Spearman r = ‒0.6112, p < 0.05) and to systolic blood pressure (Spearman r = ‒0.4746, p < 0.05) in HFrEF. CONCLUSION: Levels of sNEP were significantly reduced in HFrEF, when compared to the controls, with absent correlations to relevant HF-related features (e.g. LVEF). These findings might contribute to clarification of the diagnostic value of sNEP in HF (Tab. 2, Fig. 2, Ref. 30) Keywords: soluble neprilysin, heart failure, reduced ejection fraction, pharmacotherapy.

Impact of improving eating habits and rosmarinic acid supplementation on rat vascular and neuronal system in the metabolic syndrome model.

Decreasing high fat and high carbohydrate intake, together with the administration of natural bioactive drugs, is assumed to have a protective effect in the prevention and amelioration of the metabolic syndrome (MetS). The aim of the study was to evaluate effects of diet improvement and/or a phenolic compound (rosmarinic acid; RA) administration (100 mg/kg per d) on metabolic as well as functional changes of vessels and hippocampus caused by the MetS-like conditions. The MetS-like conditions were induced by a high-fat-fructose diet (HFFD) in Prague hereditary hypertriacylglycerolaemic (HTG) rats. The effect of diet improvement and RA administration was studied using biochemical and functional measurements. Consumption of HFFD by HTG rats resulted in the development of conditions like the MetS. The fat and fructose restriction from the diet led to amelioration of basic indicators of metabolic state in rats fed HFFD and to amendment parameters of glucose tolerance test and reduction of the IL-1ß serum levels. Moreover, aortic endothelial function was improved with an impact on blood pressure. The functional measurement of electrophysiology of the hippocampus showed that long-term potentiation of neuronal transmission course deteriorated after HFFD was improved by energy restriction. Oral administration of RA had a supporting effect not only on lipid and glucose metabolism but also on the vascular endothelium. Combination of both types of therapy induced beneficial effect on glucose tolerance and lipid peroxidation. Thus, combined improvement of diet habits and treatment with natural bioactive drugs is assumed to have protective effect in prevention and amelioration of the MetS.

Multicolour three dimensional structured illumination microscopy of immunolabeled plant microtubules and associated proteins.

BACKGROUND: In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. RESULTS: Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. CONCLUSIONS: 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

Increased expression of endothelial nitric oxide synthase and caveolin-1 in the aorta of rats with isoproterenol-induced cardiac hypertrophy.

Isoproterenol-induced cardiac hypertrophy is associated with increased expression of endothelial nitric oxide synthase in the aorta but without signs of improved endothelial function. The aim was to examine the hypothesis that increased expression of eNOS allosteric inhibitor caveolin-1 could be associated with unimproved endothelium-dependent relaxations. Rats received isoproterenol (5 mg/kg body mass, i.p., n = 13) or its vehicle (n = 14) during 1 week. Systolic blood pressure (SBP) and heart rate (HR) were measured by the tail-cuff method. Expression of eNOS and caveolin-1 was measured using immunoblotting analysis. Relaxations of isolated aorta to acetylcholine and sodium nitroprusside were evaluated ex vivo. After 1 week of isoproterenol administration, basal SBP and HR were decreased (SBP 110 +/- 3 vs. 126 +/- 3 mmHg, p < 0.05; HR 342 +/- 8 vs. 366 +/- 6 beats/min, p < 0.05). Isoproterenol increased the mass of the left ventricle (+33% +/- 4% vs. control; p < 0.05) and right ventricle (+40% +/- 9%; p < 0.05). Isoproterenol administration increased the expression of eNOS (+53% +/- 12%; p < 0.05) and caveolin-1 (+54% +/- 20%, p < 0.05) in the aorta. Relaxation of isolated aorta to acetylcholine and sodium nitroprusside showed a trend towards a worsened endothelial function and a lower sensitivity to exogenous NO. Thus, 1 week of isoproterenol administration led to increased eNOS expression in the aorta without amelioration of endothelial vasorelaxation function. Concomitant increase in caveolin-1 expression may be responsible for this paradox.

[Caveolar functions and caveolin structural proteins]. / Funkcia kaveol a ich struktúrnych proteínov kaveolínov.

Caveolae are small invaginations of the plasma membrane found in a variety of cells. The major components of caveolae are cholesterol, sfingolipids and structural proteins named caveolins. Three caveolin isoforms are known: caveolin-1, caveolin-2 and caveolin-3. Adipocytes, endothelial cells, pneumocytes and fibroblasts express caevolin-1 and-2, whereas caveolin-3 expression is limited to muscle cells types. Caveolae and caveolins have diverse functions including vesicular transport, cholesterol homeostasis, signal transduction or tumor suppression. The aim of the present review is to explain what is known about caveolae and caveolins.

Effects of amlodipine and lacidipine on cardiac remodelling and renin production in salt-loaded stroke-prone hypertensive rats.

1. Calcium channel blockers (CCBs) are anti-hypertensive drugs that are usually considered to act mainly as vasodilators. We investigated the relation between the reduction of blood pressure evoked by two long-acting CCBs and their protective effect against cardiac and renal damage in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). 2. SHRSP were exposed to high dietary salt intake (1% NaCl in drinking solution) from 8 to 14 weeks of age, with or without amlodipine or lacidipine at three dosage regimens producing similar effects on blood pressure. 3. The lowest dosages of both drugs had non-significant effects on blood pressure but inhibited the paradoxical increases in plasma renin activity (PRA) and in renin mRNA in kidney that were found in salt-loaded SHRSP. The lowest dosage of lacidipine (but not of amlodipine) restored the physiological downregulation of renin production by high salt and reduced left ventricular hypertrophy and mRNA levels of atrial natriuretic factor and transforming growth factor-beta1. 4. The intermediate dosages reduced blood pressure and PRA in a comparable manner, but cardiac hypertrophy was more reduced by lacidipine than by amlodipine. 5. Although the highest doses exhibited a further action on blood pressure, they had no additional effect on cardiac hypertrophy, and they increased PRA and kidney levels of renin mRNA even more than in the absence of drug treatment. 6. We conclude that reduction of blood pressure is not the sole mechanism involved in the prevention of cardiac remodelling by CCBs, and that protection against kidney damage and excessive renin production by low and intermediate dosages of these drugs contributes to their beneficial cardiovascular effects.

Lacidipine prevents endothelial dysfunction in salt-loaded stroke-prone hypertensive rats.

Endothelium-dependent vasorelaxation is defective in hypertensive rats, especially in conduit arteries. In the stroke-prone spontaneously hypertensive rat, impaired endothelium-dependent vasorelaxation appears to contribute to the pathogenesis of stroke independent of blood pressure. Because treatment with lacidipine, a long-acting calcium channel blocker, protects against stroke and cardiovascular remodeling in this model, we investigated the effect of this treatment on endothelium-dependent vasorelaxation in the aorta. Stroke-prone rats were exposed to a salt-rich diet (1% NaCl in drinking water) with or without lacidipine (1 mg. kg(-1). d(-1)) for 6 weeks. A high-sodium diet (1) increased systolic blood pressure, aortic weight, and wall thickness and plasma renin activity (P<0.05); (2) markedly reduced nitric oxide (NO)-mediated, endothelium-dependent relaxation of aortic rings to acetylcholine and the sensitivity to the relaxing effect of S-nitroso-N-acetylpenicillamine, an NO donor (P<0.001); and (3) induced an elevation of preproendothelin-1 mRNA levels in aortic tissue (P<0.01) without affecting endothelial NO synthase mRNA levels. Lacidipine treatment prevented the salt-dependent functional and structural alterations of the aorta, including the overexpression of the preproendothelin-1 gene, and increased endothelial NO synthase mRNA levels in aortic tissue (P<0.01). In conclusion, lacidipine protects stroke-prone hypertensive rats against the impairment of endothelium-dependent vasorelaxation evoked by a salt-rich diet, and this effect may contribute to its beneficial effect against end-organ damage and stroke.

[Inter-organ variability in enzymatic hydrolysis of ammonium salts in rats in vitro]. / Medziorgánová variabilita enzýmovej hydrolýzy organických amóniových solí u potkana in vitro.

The study evaluated the rate and kinetics of enzymatic hydrolysis of N-(2-benzoyloxyethyl)-alkyl-dimethylammonium bromides, potential easily biodegradable disinfectants of soft character. The products of enzymatic hydrolysis of the substrates under study, catalysed by microsomal esterase, included substituted substrates choline and benzoic acid which, as a hydrolytic product, was essayed by HPLC. The effect of the length of the alkyl chain of the individual homologues on the rate of enzymatic hydrolysis and their affinity to microsomal esterase of the rat liver and lung in vitro was examined. The structural modification (varying length of the aliphatic chain on the ammonium nitrogen of these compounds) was found to significantly influence the kinetics of enzymatic hydrolysis of the esteric bond. From the viewpoint of the rate of enzymatic hydrolysis no significant inter-organ variability was observed: in the liver as well as the lung the dependence of the rate of enzymatic hydrolysis on the length of the aliphatic chain possesses the shape of a falling hyperbole with the maxima for BCH2 > BCH4 > BCH8 = BCH10. The specific activity of both esterases ranges within 0.2-3.5 nmol.min-1.mg-1. From the viewpoint of affinity, a marked inter-organ difference was manifested by 10 times higher affinity of the substrates to the lung microsomal esterase in comparison with the liver one. The effect of the length of the alkyl of the individual homologues on the affinity is of a non-linear character also in this case. In both organs, a certain correlation was found between the rate of enzymatic hydrolysis and affinity to microsomal esterases.