On the localisation of d-tubocurarine in rat liver lysosomes in vivo by electron microscopy and subcellular fractionation

Summary After i.v. injection in the rat, d-tubocurarine is taken up and concentrated by the liver. A method is developed for the visualisation of d-tubocurarine inside the liver cell by electron microscopy. Glutaraldehyde fixed liver blocks were immersed in an ammonium molybdate solution; d-tubocurarine was precipitated at sites of high concentration by molybdate, to form an insoluble d-tubocurarine-molybdate complex. This precipitate was found predominantly at the surface of lysosome-like particles, but also inside these organelles. In subcellular fractionation experiments, d-tubocurarine was found with a high relative specific activity in the lysosomal fraction, lending support to a lysosomal localisation of d-tubocurarine.     

The potential role of lysosomes in tissue distribution of weak bases

The potential importance of lysosomes as a site of accumulation of weak bases in tissues is discussed. A simple mathematical treatment predicts the quantitative significance of lysosomal trapping for monoacidic and diacidic weak bases. The features which are characteristics of lysosomal trapping are discussed, particularly in comparison with active transport and intracellular binding mechanisms. These features include: linear accumulation at low concentrations; nonlinearity at higher concentrations; dependence on structural integrity of tissue; energy dependence and competition with other weak bases. Subcellular distribution studies have previously shown that weak bases accumulate extensively in membranes; however, the dependence of accumulation on the structural integrity of tissue suggests that this is not the only significant mechanism of accumulation. The results of a range of studies of tissue distribution of weak bases are discussed to illustrate that these findings are consistent with accumulation in lung and liver being attributable to a combination of lysosomal trapping and accumulation in membranes whereas, in muscle, accumulation in membranes is the predominant mechanism of accumulation. The possible pharmacokinetic significance of lysosomal trapping of weak bases is also discussed.     

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine

Dexamethasone (50 μg/kg) significantly increased the LD₅₀ of d-tubocurarine (d-TC) when administered i.p. simultaneously with d-TC. Choline (50 and 100 mg/kg) gave some protection against the lethal effects of d-TC and the cholinesterase inhibitors neostigmine (250 μg/kg) and physostigmine (1000 μg/kg) provided full protection against doses of d-TC twice the LD₅₀. The blocking effect of d-TC (75 μg/kg) on the sciatic nerve-tibialis anterior muscle preparation was antagonized by dexamethasone. Prednisolone delayed the occurrence of a complete neuromuscular block caused by d-TC in the phrenic nerve-diaphragm preparation, and antagonized the effect of d-TC on short tetanic contractions. d-TC (5 μmol/l) inhibited the [¹⁴C]choline uptake in the endplate-rich region of the rat diaphragm during stimulation. This inhibition was antagonized by dexamethasone as well as by physostigmine. The incorporation of radioactive choline into acetylcholine was inhibited in the presence of d-TC (15 μmol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect of the choline carrier system. These experimental results support the hypothesis that glucocoriticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.     

Lipid metabolism in liver of rat exposed to cadmium.

We investigated the effect of exposition to cadmium (Cd, 15ppm for 8 weeks) through drinking water on liver lipid metabolism in adult male Wistar rats. As compared to metal non-exposed (control) rats, the serum triglycerides, cholesterol and LDL+VLDL cholesterol concentrations increased. This was associated to a decrease of lipoprotein lipase activity in post heparinic plasma. The VLDL secretion from liver was not modified. Cd treatment increased triglycerides and decreased esterified cholesterol contents in liver. The high triglyceride mass was related to the increased glycerol-3-phosphate acyltransferase mRNA expression. In addition, the liver fatty acids synthesis increased, as determined by an increment of fatty acid synthetase and isocitrate dehydrogenase activities, and [(14)C]-acetate incorporation into saponifiable lipid fraction. The relative percentage of palmitic acid (16:0) and total saturated fatty acids were increased compared with control. Hepatic glucose-6-phosphate dehydrogenase, malic dehydrogenase and cholesteryl ester hydrolase activities were unchanged. In liver, the Cd treatment decreased triglyceride and cholesterol in mitochondria, also increased triglyceride in cytosol, and cholesterol and phospholipid contents in nuclei, compared with control. In addition, an increase of nuclei phosphatidylcholine synthesis was observed. Cd exposure alters directly or indirectly the serum lipid content and liver lipid metabolism.     

Late preventive effects of quinacrine on carbon tetrachloride induced liver necrosis

We have previously reported that treatments stimulating phospholipid (PL) synthesis or preventing PL degradation were late preventive agents against CCl₄-induced liver necrosis. Later studies by others postulated that stimulation of phospholipase A₂ (PLA₂) plays a role in PL degradative processes responsible for CCl₄ damage. Quinacrine (QUIN) is a well known inhibitor of PLA₂. In this work we report that QUIN (150 mg/kg i.p.) partially prevents CCl₄-induced liver necrosis at 24 h when given 30 min before or 6 or 10 h after CCl₄ (2.5 ml/kg p.o.) QUIN administration does not modify at 1 or 3 h after poisoning CCl₄ levels reaching the liver, covalent binding of CCl₄ reactive metabolites to proteins or lipids, CCl₄-induced lipid peroxidation process, CCl₄-induced decreases in body temperature, or glutathione levels in liver. QUIN concentrations in liver at times from 1 to 24 h are well over those required to inhibit PLA₂ activity. Results are compatible with the hypothesis that CCl₄ activation of PLA₂ at late stages of poisoning plays a role in CCl₄-induced liver necrosis.A. González Padrón was partially supported by a research fellowship from the Autonomic Government of the Canary Islands (Spain).     

Humanization of excretory pathway in chimeric mice with humanized liver.

The liver of a chimeric urokinase-type plasminogen activator (uPA)(+/+)/severe combined immunodeficient (SCID) mouse line recently established in Japan could be replaced by more than 80% with human hepatocytes. We previously reported that the chimeric mice with humanized liver could be useful as a human model in studies on drug metabolism and pharmacokinetics. In the present study, the humanization of an excretory pathway was investigated in the chimeric mice. Cefmetazole (CMZ) was used as a probe drug. The CMZ excretions in urine and feces were 81.0 and 5.9% of the dose, respectively, in chimeric mice and were 23.7 and 59.4% of the dose, respectively, in control uPA(-/-)/SCID mice. Because CMZ is mainly excreted in urine in humans, the excretory profile of chimeric mice was demonstrated to be similar to that of humans. In the chimeric mice, the hepatic mRNA expression of human drug transporters could be quantified. On the other hand, the hepatic mRNA expression of mouse drug transporters in the chimeric mice was significantly lower than in the control uPA(-/-)/SCID mice. In conclusion, chimeric mice exhibited a humanized profile of drug excretion, suggesting that this chimeric mouse line would be a useful animal model in excretory studies.     

Localization of mercury in CNS of the rat

The autometallographical technique has been used to determine the distribution and cellular localization of mercury deposits in the Wistar rat CNS after exposure to elemental mercury vapor (50–550 μg Hg/m³ of air for 4–24 h). In animals exposed to 50 μg Hg/m³ for 8 h, silver-enhanced mercury grains were confined to the capillary walls. Increasing the concentration of mercury to 500 μg Hg/m³ caused mercury staining to appear in neurons in the corpus striatum, mesencephalic nucleus of the trigeminal nerve and cerebellar deep nuclei. In the spinal cord, mercury appeared primarily in the motoneurons of lamina IX. Following exposure to 550 μg Hg/m³ for 12 h mercury was additionally detected in the ependyma. Animal exposure to 550 μg Hg/m³ for 24 h resulted in visible mercury deposits in the cerebellar and cerebral cortices. In the cerebral cortex, mercury was present in neurons populating lamina III in the isocortex. No mercury was detected in the allocortex. In the cerebellar cortex, mercury staining was limited to the Purkinje cells. Neurons in the thalamus contained heavy accumulations of mercury. Heavy staining for mercury was detected in lung alveolar macrophages in sections prepared from animals exposed to 550 μg Hg/m³ for 24 h. In animals exposed to 500 μg Hg/m³ or more, the primary target cells were the neurons, but glia cells also contained scattered mercury deposits. Ultrastructurally, mercury deposits were detected in the lysosomes.     

Decreased toxicity of d-tubocurarine after pretreatment with drugs elevating the intracellular level of c-AMP in mice

Summary The toxicity of d-tubocurarine in mice was slightly but significantly reduced after pretreatment with drugs which are known to increase the intracellular level of c-AMP. Papaverine, theophylline and ephedrine elevated the LD₅₀ values of d-tubocurarine by 12 to 26%. The dibutyryl derivative of c-AMP as well as c-GMP were also active in this respect.     

Pharmacokinetics and bioavailability of diclofenac in the rat

Diclofenac sodium is a widely used drug with interesting absorption and disposition features when administered to laboratory animals. The present study was undertaken to assess the pharmacokinetics of the drug after iv and gastrointestinal dosing to rats. Renal excretion of unchanged drug was negligible, but biliary excretion of the drug (unchanged and conjugated) was detected in bile duct-cannulated rats; it accounted for 27.2 and 31.2% of the total dose following iv and intraduodenal administration, respectively. Most of the drug excreted in the bile was conjugated diclofenac; unchanged drug accounted for only 4.7 and 5.4% of total diclofenac excreted in the bile after iv and intraduodenal dosing, respectively. In normal animals, intestinal absorption of the drug excreted in the bile resulted in higher drug concentrations in plasma than those obtained in bile duct-cannulated rats, but only after 60 min of dosing. When administered directly into the duodenum, diclofenac absorption was extremely fast and the maximum plasma diclofenac concentration was reached within 2 min. After oral dosing, an early peak was also observed, but it was lower than that obtained after intraduodenal dosing: 71% diclofenac hioavailability was found in bile duct-cannulated rats intraduodenally dosed, whereas in normal animals dosed by mouth a bioavailability of 79% was obtained. In normal animals intraduodenally dosed, an apparent bioavailability of 106% was observed. All of these features, particularly the influence of enterohepatic circulation on drug bioavailability, are discussed.The present work is part of a research project developed with a grant for the Plan Nacional de I + D (FAR 90-0092) of the Ministry of Industry and Energy of Spain.     

Allylamine cardiovascular toxicity: VI. Subcellular distribution in rat aortas

The cardiovascular toxin allylamine (3-aminopropene) has been shown to concentrate in elastic and muscular tissues. In this study the 14C-moiety of [14C]allylamine was traced in aortas of adult Sprague-Dawley rats after intravenously injecting 30 microCi of [14C]allylamine (spec. act. = 0.4 mCi/mM). At 5, 10, 15 and 20 min after injection 33.3-29.8% of the 14C-moiety was sequestered in aortas; at 30 min 16.8% was still present. Subcellular fractionation of the postnuclear supernatant by isopycinic centrifugation in sucrose demonstrated that 5 min after administration of [14C]allylamine, the 14C-moiety displayed a modal density peak of 1.20 g/ml. Similar activities were observed up to 30 min exposure. This modal density was similar to the distribution pattern of mitochondria based on analysis of malate dehydrogenase activities. As early as 20 min post-exposure, mitochondrial malate dehydrogenase activities of aortic mitochondria decreased, while cytosolic malate dehydrogenase activities increased, suggesting mitochondrial membrane perturbation. We suggest that the subcellular site for allylamine injury to the aorta is the mitochondrion.     

Biliary Excretion of Diazepam and its Metabolites in Man

Abstract: Ten mg diazepam was given intravenously to 12 patients with a T-tube in the common bile duct after choledochotomy (Group I) and to 10 patients after cholecystectomy (Group II). The concentrations of diazepam, of its main metabolite, N-demethyldiazepam, and of free oxazepam in the plasma of both groups and the conjugated and free concentrations of diazepam, N-demethyldiazepam, and oxazepam in the bile in the Group I were measured by gas chromatography. In Group I significantly lower plasma diazepam concentrations were obtained as compared with Group II indicating an enterohepatic circulation of diazepam. There was no significant difference in the concentrations of N-demethyldiazepam in the plasma between the two groups. In Group I the patients had frequently more free oxazepam in their plasma than in Group II. The main conjugated metabolite in the bile was N-demethyldiazepam (about 74 %). Traces of diazepam were in the conjugated form, but no conjugated oxazepam was found in the human bile. The enterohepatic circulation of diazepam and its metabolites may be partly responsible for the prolonged effects of diazepam.     

Effect of aflatoxin on rat liver lysosomes

The effect of aflatoxin on the activities of 5 rat liver lysosomal enzymes (acid deoxyribonuclease, arylsulfatases A and B, β-glucuronidase, β-glucosidase and β-galactosidase) has been investigated in vivo and in vitro. Three hours after the administration of aflatoxin, activities of most of the enzymes increased significantly reaching maximal levels at 48 hr. The activity of acid DNase increased the most (276 per cent of the control level). At the same period (48 hr) the activity of the soluble lysosomal enzymes greatly increased. Aflatoxin had a labilizing effect on lysosomal membranes, causing the release of lysosomal enzymes into the supernatant. The data are discussed in connection with the biochemical mechanism of aflatoxin intoxication.     

Pharmacokinetics of a non-narcotic analgesic,DA-5018, in rats

The pharmacokinetics of a non-narcotic analgesic, DA-5018, were compared after single intravenous (IV), subcutaneous (SC), and oral administrations, and after multiple (seven consecutive days) SC administration to rats. After IV administration of DA-5018, 1, 2, and 5 mg kg⁻¹, the pharmacokinetic parameters of DA-5018 were independent of the dose ranges studied. After oral administration of DA-5018, absorption of the drug from gastrointestinal (GI) tract was fast, but the extent of absolute bioavailability (F) was low; the values were 23.2, 23.0, and 27.3% for 2, 5, and 10 mg kg⁻¹, respectively. After single SC administration of DA-5018, absorption of the drug from the injected site was fast and the extent of absorption was fairly good; the F values were 74.5 and 71.8% for 2 and 5 mg kg⁻¹, respectively. The lower F values after oral administration of DA-5018 to rats could be due to degradation of the drug in rat GI tract and/or considerable first-pass effect. After IV, oral, and SC administration of DA-5018, the drug had a strong affinity to the rat tissues studied as reflected in the greater-than-unity tissue to plasma ratio. After IV, oral, and SC administration of the drug, the biliary and urinary excretion of unchanged DA-5018 were negligible. There was no significant difference in the pharmacokinetics or tissue distribution of DA-5018 between single and multiple SC administration of the drug, 5 mg kg⁻¹, to rats, indicating that there could be no tissue accumulation of the drug after multiple SC administration of the drug to rats. © 1998 John Wiley & Sons, Ltd.     

The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo

The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo. In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS). In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the M PS-inducing potency as compared to chloroquine carrying an isoquinoline ring system. Tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) known as a potent inducer of MPS served as reference compound. The compounds were compared at a concentration (3 μM) which did not enhance the secretion of the lysosomal enzyme β-hexosaminidase (E.C. 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell. Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms. Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine. The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system. On the other hand, quinacrine was significantly less potent than tilorone and the symmetrically substituted acridine derivative 3,6-bis[2-(diethylamino)ethoxy]acridine investigated previously. This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.     

Combined recirculation of the rat liver and kidney: Studies with enalapril and enalaprilat

Combined recirculation of the rat liver (L) and kidney (IPK) at 10 ml min^–1 per organ (LK) was developed to examine the hepatorenal handling of the precursor-metabolite pair: [¹⁴C]-enalapril and [³H]enalaprilat. Loading doses followed by constant infusion of [¹⁴C]enalapril and preformed [³H]enalaprilat to the reservoirs of the IPK or the LK preparation was used to achieve steady stale conditions. In both organs, enalapril was mostly metabolized to its dicarboxylic acid metabolite, enalaprilat, which was excreted unchanged. At steady state, the fractional excretion for [¹⁴C]enalapril (FE=0.45 to 0.48) and preformed [³H]enalaprilat (FE{pmi}=1.1) were constant and similar for both the IPK and LK. The additivity of clearance was demonstrated in the LK preparation, namely, the total clearance of enalapril was the sum of its hepatic and renal clearances. However, the apparent fractional excretion for fanned [¹⁴C]enalaprilat, FE{mi} and the apparent urinary clearance were time-dependent and higher than the corresponding values for preformed [³H]enalaprilat in both the IPK and LK. The FE{mi} and urinary clearance values further differed between the IPK and LK. Biliary clearance of formed vs. preformed enalaprilat displayed the same discrepant trends as observed for FE{mi} vs. FE{pmi} for the LK. These observations on the time-dependent and variable excretory clearance (urinary or biliary) of the formed metabolite vs. the constant, and much reduced, excretory clearance of the preformed metabolite are due to dual contributions to formed metabolite excretion: the nascently formed, intracellular metabolite which immediately underwent excretion and the formed metabolite which reentered the circulation, behaved as a preformed species. When data for the IPK and LK preparations were modeled with a physiological model with parameters previously reported for the L and IPK, all data, including metabolite excretory clearances, were well predicted. Model simulations revealed that the apparent FE{mi} differed between the LK and IPK preparations when the liver was present as an additional metabolite formation organ; the apparent excretory (urinary orGlossary k⁰ infusion rate into the reservoir - C_R reservoir concentration - C_Out,k and C_Out,L venous concentrations for the kidney and liver - C_p,k and c_P,L concentrations in renal and hepatic plasma, respectively - C_k and C_L concentrations in kidney and liver tissue, respectively - C_U and C_Bile concentrations in urine and bile, respectively - CL _b ⁱⁿ andCL _b ^ef influx and efflux clearances, respectively, at the basolateral membrane of the renal tubular cell - C _l ⁱⁿ and CL _l ^ef influx and efflux clearances, respectively, at the luminal membrane of the renal tubular cell - CL _int,K ^m renal metabolic intrinsic clearance of the drug - CL _d ⁱⁿ and CL _d ^ef influx and efflux clearances, respectively, at the sinusoidal membrane - CL _int ^m,L hepatic metabolic intrinsic clearance of the drug - CL _int,L ^b biliary intrinsic clearance - V_R plasma reservoir volume - V_P,K and V_P,L plasma volumes of the kidney and liver, respectively - V_K and V_L tissue volumes of the kidney and liver, respectively - V_U and V_Bile volumes of urine and bile, respectively - Q_K and Q_L total renal and hepatic plasma flow rates, respectively - GFR glomerular filtration rate - Q_U and Q_Bile urine and bile flow rates, respectively - f_P, f_K, and f_L unbound fractions in plasma and kidney and liver tissue, respectively This work was supported by the Medical Research Council of Canada. I. A. M. de Lannoy was a recipient of the Ontario Graduate Scholarship from the Ontario Ministry of Health; K. S. Pang was a recipient of the Faculty Development Award, Medical Research Council.     

Use of primary rat hepatocytes in the gel entrapment culture to predict in vivo biliary excretion

1. In vitro models have been widely used in characterizing the hepatobiliary elimination of compounds. However, the application of in vitro models is often limited by their imperfect simulation of in vivo situations. The current paper aims to introduce the gel entrapment culture of rat hepatocytes as an alternative method for measuring hepatobiliary transport, with the sandwich culture of rat hepatocytes set as the control.

2. First, the culture conditions of the gel-entrapped hepatocytes were modified to enhance hepatic transport function. When cultured under optimal conditions, i.e. the collagen concentration was set to 0.6?mg/mL and the regular Williams’ E medium was supplemented with epidermal growth factor, the hepatocytes maintained much higher hepatic transporter gene expression levels and transport activities than that in regular gel entrapment and sandwich culture.

3. Compared with the actual values in rats, the predicted intrinsic biliary clearance (CL_{bile,int,predicted}) of the 10 model compounds in the optimized gel entrapment culture showed a high correlation coefficient squared (R²) of 0.94, with the majority falling within the two-fold error range of the in vivo values, which was much better than the comparable sandwich culture.

4. All of these results indicate that the optimized gel entrapment culture of hepatocytes is a suitable approach for estimating in vivo biliary excretion.

     

Cactecholamines and the oedematous reaction to oestradiol in the rat uterus

The effect of catecholamines on the 4 h oedematous reaction to estradiol in the immature rat uterus was investigated. Isoprenaline was found to augment the reaction to suboptimal doses of estradiol (0.25--0.5 microgram/kg. This effect was reversed by d,l-propranolol. Adrenaline and noradrenaline were found to inhibit the reaction to oestradiol 2 microgram/kg. The effect of adrenaline was reversed by phenotalmine.     

Binding of mercury and selenium in subcellular fractions of rat liver and kidneys following separate and joint administration

The distribution of mercury and selenium has been examined in subcellular fractions of rat liver and kidneys in prolonged exposure to HgCl₂ and Na₂SeO₃ administered separately and simultaneously. The molar ratio of mercury and selenium concentrations in subcellular fractions of the organs examined varied considerably. Selenium displaced mercury from the soluble kidney fraction bound mainly with metallothionein to the nonhistone protein fraction of liver nuclei. The Hg-stimulated biosynthesis of metallothionein has been eliminated under the influence of selenium.This work was supported by the Polish-American agreement No 05-009-2, with National Institute for Occupational Safety and Health, PHS, USA