Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

关养密度和成年雄鼠填充物的气味对黄毛鼠(Rattus rattoides)的发育和性成熟的影响(英文)

黄毛鼠（Ｒａｔｔｕｓ ｒａｔｔｉｄｅｓ）在群居（４只／笼）时体重及性器官,如睾丸和附睾的增长都受到了极大的抑制;血清中睾酮含量的迅速上升以及成熟精子在附睾的曲细精管中出现的时间也比单独关养的晚。成年雄鼠的笼填充物的气味能够诱导幼年雄鼠体重,睾丸的总量和长度以及血清中睾酮的含量的增加。     

Rat metanephric organ culture in terato-embryology

The development of the permanent mammalian kidney, or metanephros, depends on mesenchymal-epithelial interactions, leading to branching morphogenesis of the ureteric bud that forms the collecting ducts and to conversion of the metanephric mesenchyme into epithelium that forms the nephrons. Rat metanephric organ culture in which these interactions are maintained is a valuable in vitro model system for investigating normal and abnormal renal organogenesis. Methods were designed to evaluate either the capacity of the ureteric bud to branch or that of the mesenchyme to form nephrons. Both are based on specific staining of the ureteric bud and the glomeruli with lectins. Using this approach, we have shown that retinoids are potent stimulating factors of nephrogenesis, acting through an increase in the branching capacity of the ureteric bud. On the other hand, several drugs such as gentamicin and cyclosporin A were found to reduce the number of nephrons formed in vitro. While gentamicin affects the early branching pattern of the ureteric bud, cyclosporin may affect the capacity of the mesencyme to convert into epithelium. This methodology therefore appears a potentially useful tool for toxicological studies new drugs.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.