Accumulation of chlorporomazine by subcellular fractions of rat brain.

1. The study of the in vitro accumulation of [3H] chlorpromazine that a state of rapid equilibrium was reached for subcellular particles from cortex, midbrain and hindbrain of rats. 2. This accumulation was not saturable by increasing the concentration of chlorpromazine, and the relationship between accumulation and concentration was not modified by temperature, preincubation with high concentration of unlabelled chlorpromazine or by lowering the fraction concentration. 3. The highest accumulation of radioactivity was seen in the midbrain region and the lowest in the hindbrain. 4. In the cortex and midbrain the membrane fraction showed the highest accumulation. 5. The effect of pH on the accumulation indicated that a significnat rise was seen between pH 8 and 5. 6. This factor could also be explained in terms of solubility of the drug in membranes. 7. Studies on the metabolic fate of the drug during incubation indicated that there was a fairly rapid breakdown during incubation, the highest being seen with mitochondrail fractions and the least with membrane procedures. 9. It is concluded that accumulation of chlorpromazine by subcellular fractions in vitro may be associated with solubility in membranes and that the picture may be complicated by the presence of breakdown products.     

Identification of the nicotinic and muscarinic acetylcholine receptors in subcellular fractions of mouse brain

Binding of radioactive cholinergic ligands was assayed by equilibrium dialysis to mitochondrial and synaptosomal preparations from mouse brain. Whereas ³H-atropine bound to two sites. ³H-nicotine. ³H-pilocarpine and ³H-acetylcholine (ACh) (after inhibition of cholinesterases in the tissue preparation with pyridostigmine) bound reversibly to single sites with high affinities (7·2, 8·1 and 23 nm. respectively). The ³H-nicotine binding sites (3·1 pmol/g brain) were inhibited by nicotinic but not muscarinic drugs, and the reverse effect was on the ³H-pilocarpine binding sites (3·7 pmol/g brain). The ³H-ACh binding sites were inhibited by both nicotinic and muscarinic drugs and α-bungarotoxin. and equalled approximately the total concentration of the nicotinic and muscarinic sites.It is suggested that the ACh binding sites are on the ACh receptor (AChR), which is found at concentrations similar to acetylcholinesterase in the mouse brain. These AChR sites are divided almost equally between muscarinic and nicotinic types. The data also suggest that most of the binding sites for ³H-atropine or ¹²⁵I-α-bungarotoxin in the mouse brain are not on AChR.     

gamma-Aminobutyric acid in subcellular fractions of mouse brain and its relation to convulsions.

The in vivo effects of convulsant drugs (penicillamine, semicarbazide and isonicotinic acid hydrazide) on the γ-aminobutyric acid (GABA) levels in subcellular fractions were studied in mice. All the drugs reduced the concentration of GABA in the synaptosomal fraction; it was unlikely that this decrease in the GABA concentration was due to redistribution or degradation during the separating procedures. When the convulsions were prevented by simultaneous pyridoxine injection, the GABA level in synaptosomes rose to normal. It is suggested that the decrease of GABA concentration in nerve endings may be an important factor in the onset of some kinds of convulsions.     

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions

1. The oxidative metabolism of ¹⁴C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by?h.p.l.c. employing continuous radioactivity monitoring of the column effluent.

2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per?mg protein, 2-pyridone (2PO) at 207 pmol/min per?mg and 4-pyridone (4PO) at 154 pmol/min per?mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites.

3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate.

4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism.

5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per?mg protein, 2PO at 17 pmol/min per?mg and 4PO at 61 pmol/min per?mg.

6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.     

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions

1. The oxidative metabolism of 14C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by h.p.l.c. employing continuous radioactivity monitoring of the column effluent. 2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per mg protein, 2-pyridone (2PO) at 207 pmol/min per mg and 4-pyridone (4PO) at 154 pmol/min per mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites. 3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate. 4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism. 5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per mg protein, 2PO at 17 pmol/min per mg and 4PO at 61 pmol/min per mg. 6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.     

The metabolism of 1,3-dinitrobenzene by rat testicular subcellular fractions.

The metabolism of 1,3-dinitrobenzene by rat testicular subcellular fractions (microsomes, cytosol or 9000 x g supernatant (S-9)) was studied. The effects of NADPH, oxygen, glutathione (GSH) and carbon monoxide upon the rate of metabolism were determined. Three metabolites were identified, and characterised as 3-nitrosonitrobenzene, 3-nitrophenylhydroxylamine and 3-nitroaniline by co-chromatography with authentic standards. These findings indicate the presence of an inherent enzyme system capable of facilitating nitro-reduction in the testes. The implications of these findings to the mechanism of 1,3-DNB-induced testicular toxicity are discussed.     

Effect of choline acetyltransferase inhibitors on mouse and guinea-pig brain choline and acetylcholine

These experiments measure the effect of two choline acetyltransferase (CAT) inhibitors, viz. 4-(1-naphthylvinyl) pyridine (NVP) and 4-(3-chlorophenylvinyl) pyridine (3'-chloro-4-stilbazole; CS), on mouse and guinea-pig brain acetylcholine (ACh) and choline. The intraperitoneal administration of NVP or CS appeared to inhibit CAT partially in both species, but both compounds were without effect on steadystate levels of ACh. In the mouse CS, but not NVP, increased brain choline. Both CS and NVP were shown to reduce, but not totally inhibit, the synthesis of mouse brain ACh. The inability of these compounds to decrease steady state levels, while apparently decreasing synthesis rates, suggests that CAT may not be the rate-limiting step in ACh synthesis.     

Inhibitory effects of chronic administration of morphine on uridine and thymidine incorporating abilities of mouse liver and brain subcellular fractions

Attempts have been made to examine the in vitro and in vivo effects of morphine sulfate on the incorporation of ¹⁴C-labeled uridine and thymidine into acid-insoluble fractions of mouse liver and brain. Morphine, in vitro or after administration in vivo, produced no significant changes in the uridine and thymidine incorporating abilities of liver and brain homogenates. The chronic administration of morphine produced dose-dependent decreases in uridine and thymidine incorporating abilities of liver and brain homogenates and of their subcellular fractions such as nuclei, mitochondria and microsomes.     

Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish (Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.     

Effect of diazepam on mouse whole brain and brain area acetylcholine and choline levels

A single i.v. dose of diazepam, 5 mg/kg, increased mouse whole brain acetylcholine levels. Choline levels, choline acetyltransferase and acetylcholinesterase activities were not affected, which is consistent with the hypothesis that diazepam blocks release of acetylcholine. Diazepam increased acetylcholine levels in the hemispheres and diencephalon but not in the cerebellum or mesencephalon. The effect lasted for 4 hr in the hemispheres and for 30 min in the diencephalon. This short-lastig biochemical action precludes a correlation with the long-lasting action of diazepam against pentylenetetrazole.     

Effects of ischemia and pharmacological treatment on subcellular fractions from neonatal rat brain

The effects of complete ischemia and of in vivo pharmacological treatment with trimetazidine were studied on some enzymatic activities related to energy transduction: lactate dehydrogenase for anaerobic glycolysis; citrate synthase and malate dehydrogenase for the Krebs' cycle; total NADH-cytochrome c reductase and cytochrome oxidase for the electron transport chain; glutamate dehydrogenase for amino acid metabolism and acetylcholine esterase for acetylcholine metabolism. These enzymatic activities were evaluated in brains of 10-day-old rats, at three different subcellular levels: homogenate in toto, purified mitochondrial fraction, crude, synaptosomal fraction. Complete normothermic post-decapitative ischemia of 30 min duration increased the activity of cytochrome oxidase in the homogenate in toto and increased the activities of citrate synthase and malate dehydrogenase in the purified mitochondrial fraction, the activities of the enzymes evaluated in the crude synaptosomal fraction being unaffected. The i.p. treatment with trimetazidine (at the dose level of 50 mg . kg-1) was without any significant effect on the tested enzymatic activities.     

Comparison of the enzymatic and colchicine binding properties of subcellular fractions of rat brain

Capacity for colchicine binding and activities of lactate dehydrogenase, monoamine oxidase and cholinesterase were determined in subfractions of adult rat brain. Colchicine binding activity was mainly found in cytoplasmic fractions. The fractions containing membranes bound little colchicine. Activity and specific activity of colchicine binding were roughly comparable with the corresponding values of lactate dehydrogenase. Colchicine binding in particulate fractions therefore is probably due to contamination with cytoplasmic protein. Polyacrylamide gel electrophoresis revealed some similarity between the protein patterns of the subsynaptosomal particulate fractions and showed that colchicine binding protein is not present in synaptic vesicles.     

In vitro febantel transformation by sheep and cattle ruminal fluids and metabolism by hepatic subcellular fractions from different animal species

Febantel and one of its main metabolites, febantel sulphoxide, are chemically modified to only a slight extent when incubated in vitro with sheep and cattle ruminal fluids; other major metabolites, fenbendazole and oxfendazole, are respectively, oxidized to oxfendazole and reduced to fenbendazole. Febantel is negligibly metabolized by hepatic cytosol fractions but microsome preparations effect more extensive metabolic transformations. Important differences in this respect were found between microsome preparations from rat, horse, pig, cattle, sheep, chicken and trout livers.