Amino acid concentrations and protein metabolism of two types of rat skeletal muscle in postprandial state and after brief starvation.

We have investigated amino acid concentrations and protein metabolism in musculus extensor digitorum longus (EDL, fast-twitch, white muscle) and musculus soleus (SOL, slow-twitch, red muscle) of rats sacrificed in the fed state or after one day of starvation. Fractional protein synthesis rates (FRPS) were measured using the flooding dose method (L-[3,4,5-3H]phenylalanine). Activities of two major proteolytic systems in muscle (the ubiquitin-proteasome and lysosomal) were examined by measurement of chymotrypsin like activity of proteasome (CTLA), expression of ubiquitin ligases atrogin-1 and muscle-ring-finger-1 (MuRF-1), and cathepsin B and L activities. Intramuscular concentrations of the most of non-essential amino acids, FRPS, CTLA and cathepsin B and L activities were in postprandial state higher in SOL when compared with EDL. The differences in atrogin-1 and MuRF-1 expression were insignificant. Starvation decreased concentrations of a number of amino acids and increased concentrations of valine, leucine, and isoleucine in blood plasma. Starvation also decreased intramuscular concentrations of a number of amino acids differently in EDL and SOL, decreased protein synthesis (by 31 % in SOL and 47 % in EDL), and increased expression of atrogin-1 and MuRF-1 in EDL. The effect of starvation on CTLA and cathepsin B and L activities was insignificant. It is concluded that slow-twitch (red) muscles have higher rates of protein turnover and may adapt better to brief starvation when compared to fast-twitch (white) muscles. This phenomenon may play a role in more pronounced atrophy of white muscles in aging and muscle wasting disorders.

Pharmacokinetics of boldine in control and Mrp2-deficient rats.

The aim of the present study was to describe the currently poorly understood pharmacokinetics (PK) of boldine in control rats (LW, Lewis rats), and Mrp2 transporter-deficient rats (TR(-)). Animals from the LW and TR(-) groups underwent a bolus dose study with 10 mg/kg of boldine applied either orally or intravenously in order to evaluate the major PK parameters. The TR(-) rats demonstrated significantly reduced total clearance with prolonged biological half-life (LW 12+/-4.6 versus TR(-) 20+/-4.4 min), decreased volume of distribution (LW 3.2+/-0.4 l/kg versus TR(-) 2.4+/-0.4 l/kg) and reduced bioavailability (LW 7 % versus TR(-) 4.5 %). Another set of LW and TR(-) rats were used for a clearance study with continuous intravenous administration of boldine. The LW rats showed that biliary and renal clearance formed less than 2 % of the total clearance of boldine. The treatment of samples with beta-glucuronidase showed at least a 38 % contribution of conjugation reactions to the overall clearance of boldine. The TR(-) rats demonstrated reduced biliary clearance of boldine and its conjugates, which was partly compensated by their increased renal clearance. In conclusion, this study presents the PK parameters of boldine and shows the importance of the Mrp2 transporter and conjugation reactions in the elimination of the compound.

Boldine attenuates cholestasis associated with nonalcoholic fatty liver disease in hereditary hypertriglyceridemic rats fed by high-sucrose diet.

The aim of the current study was to clarify the effect of high sucrose diet (HSD) on bile formation (BF) in rats with hereditary hypertriglyceridemia (HHTg). Potentially positive effects were studied for boldine, a natural choleretic agent. Administration of HSD to HHTg rats led to increased triglyceride deposition in the liver. HSD reduced BF as a consequence of decreased biliary secretion of bile acids (BA) and glutathione. Responsible mechanism was down-regulation of hepatic transporters for BA and glutathione, Bsep and Mrp2, respectively. Moreover, gene expressions of transporters for other constituents of bile, namely Abcg5/8 for cholesterol, Abcb4 for phospholipids, and Oatp1a4 for xenobiotics, were also reduced by HSD. Boldine partially attenuated cholestatic effect of HSD by promotion of biliary secretion of BA through up-regulation of Bsep and Ntcp, and by increase in biliary secretion of glutathione as a consequence of its increased hepatic disposition. This study demonstrates mechanisms of impaired BF during nonalcoholic fatty liver disease induced by HSD. Altered function of responsible transporters suggests also potential for changes in kinetics of drugs, which may complicate pharmacotherapy in subjects with high intake of sucrose, and with fatty liver disease. Sucrose induced alterations in BF may be alleviated by administration of boldine.

Collagenolytic potential of rat liver myofibroblasts.

Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent alpha-smooth muscle (alpha-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in alpha-SMA and beta-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-beta family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.

Low expression of NQO1 predicts pathological complete response to neoadjuvant chemotherapy in breast cancer patients treated with TAC regimen.

The aim of this study was to evaluate preoperative tumour expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) along with other biological markers as potential predictors of pathological complete response (pCR) to neoadjuvant docetaxel, doxorubicin, and cyclophosphamide-containing (TAC) chemotherapy in patients with primary breast cancer. Sixty-one patients who received neoadjuvant chemotherapy (NCT) with TAC regimen were enrolled in this prospective study. The pre- and post- NCT expression of oestrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor 1 and 2 (EGFR and HER2), NQO1, Ki-67 proliferation index, multidrug resistance protein 1 (MDR1), p53 and BCL2 were evaluated by immunohistochemistry. The pCR was reached in 14 patients (23 % of the study group). Multivariate analysis demonstrated that patients with ER-, PR-, NQO1- negative, and Ki-67-positive tumours had a significantly higher chance to achieve pCR. Within the biological subtypes, the highest pCR rate (50 %) was seen in triple-negative (i.e. ER-, PR-, HER2-) tumours. Post-operative evaluation showed that in comparison to pre-operative tissue samples, NQO1 expression was significantly increased, while Ki-67 and HER2 decreased, in the residual tissue after NCT. In conclusion, the present data suggests that NQO1 expression may be a novel diagnostic biomarker for the prediction of positive response to NCT in patients with breast cancer.

Predictive biomarkers in breast cancer: their value in neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NCT) of breast cancer enabled improved outcomes especially in patients with advanced and inflammatory diseases. Biological heterogeneity of these tumors, however, requires better molecular characterization of the malignant tissue with consequent individualization in the selection of appropriate agents. To date, numerous molecular markers have been identified, and some of them (e.g., measurement of hormonal or growth factors receptors) are already routinely used for breast cancer classification before NCT. In the present article, we summarize current knowledge about established as well as promising biomarkers which have demonstrated prognostic or predictive value in NCT of breast cancer.

Role of transplanted bone marrow cells in response to skeletal muscle injury.

The recently discovered capacity of bone marrow cells (BMCs) to contribute to injury-induced skeletal muscle regeneration has brought new possibilities in the treatment of skeletal muscle diseases. However, a suitable method of BMC transplantation usable for such therapy has to be established. In this work, recipient mice were intramuscularly injected with cardiotoxin, then whole-body lethally irradiated to eradicate satellite cells in their injured tibialis anterior (TA) muscles and to suppress haematopoiesis, and subsequently intravenously transplanted with lacZ+ BMCs with the aim to investigate the role of exogenous BMCs in response to skeletal muscle injury. Seven to 33 days after grafting, recipient TA muscles were examined to detect donor-derived X-gal+ cells and analysed by quantitative PCR. In injured recipients' muscles, X-gal positivity was identified 14 and 33 days after grafting in some infiltrating neutrophils and macrophages, infrequently in fibroblasts of endomysium, and in many large multinucleated cells (devoid of myogenic markers desmin and nestin) resembling foreign body giant cells situated in the vicinity of necrotic muscle fibres. qPCR confirmed the presence of transplanted lacZ+ BMCs in injured recipients' muscles. Our results proved the ability of intravenously transplanted adult BMCs to settle in injured muscles and generate blood cells that infiltrated endomysium and took part in the cleaning reaction. After inhibition of endogenous myogenesis, BMCs were not able to participate in formation of new muscle fibres due to persisting necrosis of degenerated muscle fibres. Instead, BMCs attempted to resorb necrotic structures, which confirmed the indispensable role of bone marrow-derived macrophages in skeletal muscle regeneration.

Endoglin co-expression with eNOS, SMAD2 and phosphorylated SMAD2/3 in normocholesterolemic and hypercholesterolemic mice: an immunohistochemical study.

Endoglin, a homodimeric transmembrane glycoprotein, is a part of the transforming growth factor-beta (TGF-beta) receptor cascade. It has been demonstrated that endoglin can affect TGF-beta signaling and eNOS expression by affecting SMAD proteins in vitro. We planned to go one step forward and evaluate whether endoglin is co-expressed with SMAD2, phosphorylated SMAD2/3 protein and eNOS in endothelium of normocholesterolemic C57BL/6J mice, and in advanced atherosclerotic lesions in hypercholesterolemic apoE/LDLr-deficient mice by means of fluorescence immunohistochemistry. Female C57BL/6J mice were fed with a chow diet (standard laboratory diet) for 12 weeks after weaning (at the age of 4 weeks). Two-month-old female apoE/LDLr-deficient mice were fed the western type diet (atherogenic diet) containing 21% fat (11% saturated fat) and 0.15% cholesterol for 2 months. Immunohistochemical analysis of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS expression was performed in mice aortic sinus. Immunohistochemical analysis showed the expression of endoglin in intact endothelium in both C57BL/6J and apoE/LDLr-deficient mice and in endothelium covering the atherosclerotic lesion in apoE/LDLr-deficient mice. Fluorescence immunohistochemistry revealed co-expression of endoglin with SMAD2, phosphorylated SMAD2/3 and eNOS in intact aortic endothelium in C57BL/6J mice. Moreover, strong co-localization of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS was also detected in endothelium covering atherosclerotic lesions in apoE/LDLr-deficient mice. In conclusion, we suggest that endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS may be important in vessel endothelium homeostasis underlying their role in atherogenesis.

Diurnal variation of 6beta-hydroxycortisol in cardiac patients.

The 24-hour urinary excretion of 6-beta-hydroxycortisol (6beta-OHC) and the urinary ratio of 6beta- hydroxycortisol/cortisol (6beta-OHC/UFC) have been proposed as noninvasive probes for human cytochrome P450 3A4 isoform (CYP3A4). In this study, we evaluated within- and between-day variability of 6beta-OHC excretion and 6beta-OHC/UFC ratio in nine Caucasian men with cardiac disease. Each study participant was asked to collect 24-hour urine specimens during four consecutive days in five standardized time intervals. Concentrations of UFC and 6beta-OHC were determined by immunoassay and the high-performance liquid chromatographic (HPLC) method, respectively. The HPLC method was accurate and precise, as indicated by the recovery rate of 96.5-103.3 % and less than 5.2 % and 6.3 % of the coefficient of variation for within-run and between-run assay, respectively. In patients, diurnal variations in UFC and 6beta-OHC excretion were parallel. Consequently, 6beta-OHC/UFC ratio remained stable during the day. Both, 6beta-OHC excretion and 6beta-OHC/UFC ratio showed significant relationship between 24-hour value and values measured in corresponding collection periods with best correlations obtained from night interval (22.00-06.00, r = 0.86-0.91). These results indicated that urinary 6beta-OHC excretion and 6beta-OHC/UFC ratio measured in overnight/morning urine could precisely reflect 24-hour values even in severely ill patients. In addition, a simple and sensitive HPLC method was described for determination of 6beta-OHC in urine.

Differential effects of statins and alendronate on cholinesterases in serum and brain of rats.

Acetylcholinesterase (AChE) inhibitors represent standard treatment of Alzheimer's disease. Cholesterol plays an important role in Alzheimer's disease development. Because cholesterol synthesis may be inhibited by statins or bisphosphonates, we hypothesized that these drugs might possibly have an influence on cholinesterases. Moreover, we also evaluated if the cholesterol-lowering agents that cross the blood-brain barrier (e.g. simvastatin) should be more effective than those which do not (e.g. atorvastatin). Four groups of rats were orally administered simvastatin, atorvastatin, alendronate or vehicle for seven days. Thereafter, blood samples were taken and the basal ganglia, septum, frontal cortex, and hippocampus were isolated from brains for measurement of acetylcholinesterase activity. In the blood, activities of neither acetyl- nor butyrylcholinesterase were influenced by any of the applied drugs. In the brain, no significant changes in AChE activity were observed after administration of atorvastatin. Both simvastatin and alendronate significantly suppressed the activity of AChE in the frontal cortex. In conclusion, our results confirmed the hypothesis that cholesterol-modifying drugs modulate AChE activity and it is more reasonable to use a blood-brain barrier penetrating drug.

Expression of MRP2 and MDR1 transporters and other hepatic markers in rat and human liver and in WRL 68 cell line.

Here we describe a comparative study of phenotypic properties of hepatic cells in situ and in vitro. We analyzed the expression levels and distribution patterns of ABC transporters MRP2 and MDR1, pan-cytokeratin, cytokeratin 18, albumin, alpha-fetoprotein and the specific hepatocyte marker OCH1E5 in the fetal and adult rat as well as human liver tissue and in human fetal hepatocytes of WRL 68 cell line using peroxidase immunohistochemistry or immunofluorescence. Transporters MRP2 and MDR1 were expressed in all examined liver tissues, except rat ED13 embryo. The immunopositivity of these proteins was localized to the canalicular membrane of differentiating and mature hepatocytes but in the later developmental stages and in the adult liver tissues it was also found in the apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunoreactivity appeared after 5-6 days of cultivation and both transporters were fully expressed in the plasmalemma and in the cytoplasm 9 days after the passage. In conclusion, we observed only moderate variances reflecting diverse ontogenetic phases between the fetal and adult liver tissue. To study functions of hepatocytes in vitro, WRL 68 cells have to differentiate prior to the examination. Our findings indicate that WRL 68 cells can undergo differentiation in vitro and their antigenic profile closely resembles hepatocytes in the human liver.

Influence of amiodarone on urinary excretion of 6beta-hydroxycortisol in humans.

The present study was undertaken to evaluate the use of cortisol 6beta-hydroxylation in defining the effect of amiodarone on cytochrome CYP3A activity. To accomplish this goal, the in vivo activity of CYP3A was estimated by measuring the 24-hour urinary excretion of 6beta-hydroxycortisol (6beta-OHC) and by calculating 24-hour ratio of 6beta-hydroxycortisol to urinary free cortisol (6beta-OHC/UFC ratio). Nine cardiac patients scheduled for amiodarone treatment were recruited to participate in this study. Urine was collected over a 24-hour period from each subject before the first amiodarone administration and during the third day of oral administration of amiodarone (200 mg four times daily as a loading dose). Three days of amiodarone treatment caused a significant decrease (p<0.05) in both the 6beta-OHC/UFC ratio and the 24-hour urinary excretion of 6beta3-OHC. These results suggest that amiodarone is an inhibitor of CYP3A activity.

Influence of CYP3A metabolizer status on the pharmacokinetics and pharmacodynamics of amiodarone.

UNLABELLED: The present work was designed to determine whether the individual differences in pharmacokinetics and pharmacodynamics of amiodarone and its N-desethyl metabolite are related to cytochrome CYP3A metabolizer status. METHODS: 12 cardiac patients with inducible ventricular tachyarrhythmias during the baseline electrophysiologic study were enrolled in this study. Urinary 24-hour excretion of 6 beta-hydroxycortisol (6 beta-OHC and the ratio of 6 beta-hydroxycortisol to urinary free cortisol (6 beta-OHC/UFC) were measured before the first amiodarone administration. Trough plasma concentrations of amiodarone and N-desethylamiodarone (N-DEA) were measured after 79 +/- 11 days (2nd period) and after 182 +/- 25 days (3rd period). Electrophysiologic effects of amiodarone therapy were established with serial electrophysiologic studies in 9 of these patients at the baseline and after 79 +/- 11 days (during the second period). RESULTS: Both the 6 beta-OHC excretion and 6 beta-OHC/UFC ratio varied approximately 6-fold between the patients. We found significant inverse correlation between the 6 beta-OHC excretion and the trough plasma concentrations of amiodarone at the time of the 3rd period (rs = -0.58, p < 0.05). Similarly, there was correlation between the 24-hour urinary 6 beta-OHC excretion and trough plasma concentrations of amiodarone during the 3rd period (rs = -0.64, p < 0.025). We were unable to detect any association between CYP3A activity and amiodarone pharmacodynamics. CONCLUSION: This study points toward important information value of CYP3A metabolizer status in the context of therapeutic drug monitoring of amiodarone.