Studies on NADPH-dependent chloral hydrate reducing enzymes in rat liver cytosol

Chloral hydrate, a sedative hypnotic and also a major metabolite of trichloroethylene in higher animals, is reduced to trichloroethanol by liver extracts. The reducing activity in rat liver cytosol could be separated into four fractions [one NADH- (F₁) and three NADPH-dependent (F₂, F₃ and F₄)] by DEAE-cellulose column chromatography. By several procedures, F₂ was purified over 1000-fold and F₄ was purified over 600-fold from liver cytosol. As judged from polyacrylamide gel electrophoresis performed with and without the addition of sodium dodecylsulfate, the final preparations were essentially homogeneous. They differed in molecular weight, mobility on polyacrylamide gel electrophoresis, pH optimum, substrate specificity, and sensitivity to inhibitors. The molecular weights were estimated to be 36,000 and 32,500 for F₂ and F₄, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The estimation of molecular weights by thin-layer gel chromatography indicated that the enzymes were monomers. An examination of over thirty substrates revealed that both enzymes catalyzed the reduction of long-chain aliphatic, alicyclic and aromatic aldehydes as well as halogenated acetaldehyde. The F₂ enzyme acted on d-glucuronate, indicating that it was identical to the aldehyde reductase recently reported by other workers (l-gulonate: NADP⁺ 1-oxidoreductase EC 1.1.1.19). The F₄ enzyme, on the other hand, preferentially acted on C₂₄ 3-ketosteroids.     

The effects of some mitochondrial translocase inhibitors on the activity of rat liver transhydrogenase

Non-energy dependent transhydrogenase activity in submitochondrial particle is stimulated at pH 8 by some inhibitors of mitochondrial carboxylate translocases. On solubilising the enzyme or assaying it at pH 6 these inhibitors have little effect. K_m values remain unchanged under all these conditions. Conversely, energy-dependent transhydrogenase activity is inhibited by these substances possibly due to their direct action on proton transport. It is suggested that the observed effects may all be due to changes in membrane conformation induced by the inhibitors.     

Metabolism of malondialdehyde by rat liver aldehyde dehydrogenase

Mammalian liver contains a group of pyridine nucleotide linked aldehyde dehydrogenases [E.C. 1.2.1.3] which are present in high specific activity and possess wide substrate specificities. Malondialdehyde (MDA), a difunctional three-carbon aldehyde thought to be toxic, is generated during membrane lipid peroxidation in hepatocytes. The role of aldehyde dehydrogenase (ALDH) in the metabolism of MDA was tested in vitro with subcellular fractions and semipurified cytosolic preparations from rat livers. The cytosolic fraction accounted for virtually all of the MDA (50 microM) metabolizing activity observed in the postnuclear supernatant fraction. The rate of MDA disappearance was relatively low in the mitochondrial fraction and was not detectable in reaction mixtures which contained microsomes. Rat liver cytosol contained two ALDHs with MDA metabolizing activity. These enzymes were separated by DEAE-cellulose ion exchange chromatography and had apparent Km values of 16 microM and 128 microM for malondialdehyde. Mitochondria contained an ALDH enzyme with lower affinity (Km of 7.3 mM with NAD+) for malondialdehyde. These data show that rat liver contains at least three ALDH enzymes which oxidize malondialdehyde.     

Effects of lithium on the activities of phosphofructokinase and phosphoglucomutase and on glucose-1,6-diphosphate levels in rat muscles, brain and liver

Incubation of rat diaphragm muscle in the presence of lithium chloride (a drug used widely in the therapy of patients with mental illness), resulted in a sharp decrease in the level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism. This decrease in Glc-1,6-P2, the most potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked reduction in the activities of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. A decrease in Glc-1,6-P2 and the concomitant reduction in the activities of phosphofructokinase and phosphoglucomutase, were also obtained in the rat gastrocnemius and tibialis anterior muscles, as well as in brain, following Li+ injection. In contrast to its effects in muscles and brain, Li+ did not exert any effect on Glc-1,6-P2 level and on the enzymes' activities in the liver. The marked inhibition of brain and muscles phosphofructokinase (the rate-limiting enzyme in glycolysis) induced by Li+, may play an important role in the mechanism of the therapeutic action of this agent in the manic state.     

Inhibition of the enzymes of glutathione metabolism by mercuric chloride in the rat kidney: reversal by selenium

The treatment of rats with 10 mumoles/kg (s.c.) of mercuric chloride (Hg2+) caused time-dependent decreases in the activities of the enzymes of the glutathione (GSH) metabolism pathway in the kidney. Twenty-four hours after administration of Hg2+, the activities of gamma-glutamylcysteine synthetase and glutathione disulfide (GSSG)-reductase in the kidney were decreased by 50-60%, and the activities of the GSH catabolic enzymes, gamma-glutamyl transpeptidase and GSH-peroxidase, were decreased by 25-35%. In the liver, only the activity of GSSG-reductase was decreased at this time. The observed decreases in the enzyme activities were not accompanied by a depression in the cellular protein concentration. The same pattern of enzyme response was noted when rats were given 30 mumoles/kg Hg2+; however, the decreases in the specific activity of the enzymes were accompanied by great losses in the cellular protein concentrations in both the liver and the kidney (35-40%). This dose of Hg2+ also caused significant decreases in the concentration of GSH in both organs. In vitro, Hg2+ only inhibited the activity of GSSG-reductase. When rats were given sodium selenite (Na2SeO3; 5, 10 or 20 mumoles/kg, s.c.) 30 min after Hg2+ treatment (10 mumoles/kg), the Hg2+-related depressions in the activities of the enzymes of GSH metabolism in the liver and the kidney were blocked. Also, in rats treated with 30 mumoles/kg Hg2+, the administration of 10 mumoles/kg selenium significantly decreased the magnitude of depression in the concentration of GSH in the kidney.     

Interaction between ethanol and carbohydrate on the metabolism in rat liver of aromatic and chlorinated hydrocarbons

Metabolic rates of eight hydrocarbons (benzene, toluene, styrene, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethylene, and trichloroethylene) were measured in vitro with the livers from rats that had consumed ethanol for 3 weeks in combination with various diets. Ethanol and carbohydrate antagonized each other in connection with activity of hepatic drug-metabolizing enzymes (mixed-function oxidases); the former increased and the latter decreased these enzymes. A decrease (increase) in carbohydrate intake augmented (suppressed) the action of ethanol in a dose-related manner. In particular, a combination of ethanol with a low-carbohydrate diet (DeCarli and Lieber diet) enhanced enzyme activity and potentiated carbon tetrachloride-induced hepatotoxicity. Enhancement of metabolism and potentiation of toxicity were due to the combination rather than to ethanol per se.     

Effects of copper on the binding of agonists and antagonists to muscarinic receptors in rat brain

Studies were performed to assess the effects of copper treatment in vitro on muscarinic binding parameters in rat brain homogenates. Brainstem, an area low in copper, was found to be insensitive to copper treatment as compared to forebrain, a region of relatively high copper content. Inclusion of 3 microM copper in forebrain homogenates decreased the number of sites seen by [3H]-l-quinuclidinyl benzilate (QNB) by 40-50%. Copper-enhanced displacement of bound QNB was noted for agonists and antagonists. Both ligands showed maximal effects at 6 microM copper, although quantitative differences could be determined at any copper level. At levels of maximal effect, the increase in QNB displacement was greater than or less than 50% for agonists and antagonists respectively. Two-site analyses of carbamylcholine (CCH) binding showed that the addition of 1 microM copper to forebrain homogenates increased the percentage of high affinity sites (alpha) from 42 to 70%. The IC50 decreased from 3.1 to 1.7 microM, but the dissociation constants for the high and low affinity sites were not changed. The effect of added copper on CCH binding to muscarinic receptors was reversible with the addition of the copper-chelating agent triethylene tetramine.     

Effects of chronic dietary lithium on activity and regulation of (Na+,K+)-adenosine triphosphatase in rat brain

Chronic dietary administration of lithium appeared to reduce (Na⁺,K⁺)-adenosine triphosphatase in rat brain by at least two mechanisms. Selective inhibition of the enzyme form with high affinity for ouabain occurred in hippocampus. Nonselective inhibition, manifested by decreased V_max for activation by Na⁺. occurred in hippocampus, cortex and corpus striatum. There was no effect on Na⁺ affinity. Because lithium administration decreased shock-elicited fighting behavior in the presence of moderate tissue lithium levels and in the absence of changes in weight or gross appearance or behavior, the effects on adenosine triphosphatase were pharmacologic rather than toxic.     

Inhibition of drug metabolism by the antimalarial drugs chloroquine and primaquine in the rat

The effect of the antimalarial drugs chloroquine (CQ) and primaquine (PQ) on rat liver microsomal drug metabolism has been studied in vitro and in vivo. After acute administration, PQ increased pentobarbitone sleeping time in a dose-related manner [control, 94.0 ± 9.4 min; 10mg/kg, 137.0 ± 2.4 min; 20mg/kg, 197.0 ± 7.5 min; 50 mg/kg, 269.0 ± 2.9 min (means ± S.E.M.)], prolonged zoxazolamine paralysis time (control, 140.0 ± 10.0 min; 50 mg/kg, 341.5 ± 25.6 min) and decreased antipyrine blood clearance from 2.17 ± 0.19 to 0.86 ± 0.12 ml/min. CQ showed no effect on pentobarbitone sleeping time or zoxazolamine paralysis time, but decreased antipyrine clearance from 2.17 ± 0.19 to 1.11 ± 0.18 ml/min. Both drugs inhibited aminopyrine N-demethylase activity, although the concentration required to produce 50% inhibition was much greater for CQ (10 mM) than for PQ (approximately 0.1 mM). Lineweaver-Burk plots showed that CQ inhibited competitively whereas PQ inhibition was apparently non-competitive. Ethoxyresorufin O-deethylase activity decreased by about 40 and 50% in the presence of CQ and PQ respectively (250 nM, equimolar with substrate). There was no evidence of induction following chronic administration of CQ and PQ (50 mg/kg/day for 4 days). There was an apparent decrease in cytochrome P-450 content and aminopyrine N-demethylase activity was decreased. These results demonstrate that PQ and CQ inhibit hepatic drug metabolism both in vitro and in vivo and that PQ appears to be the more potent inhibitor.     

Influence of hypoxia on the metabolism and excretion of misonidazole by the isolated perfused rat liver--a model system

The isolated perfused rat liver was evaluated as a model system for the characterization of misonidazole metabolism under hypoxic conditions. Misonidazole metabolism by livers perfused under aerobic conditions was also examined. The clearance of misonidazole was more than three times greater under anaerobic compared to aerobic conditions (4.94 +/- 1.56 vs 1.27 +/- 0.22 ml/min; means +/- S.D., N = 3). Misonidazole metabolites were detected only in the bile. Analysis of these metabolites by reverse-phase high performance liquid chromatography (HPLC) demonstrated that misonidazole metabolism was also qualitatively changed when anaerobic conditions were employed. Misonidazole beta-glucuronide was the major metabolite detected under aerobic conditions, but it was a minor metabolite in anaerobically perfused livers. The three major metabolites produced under anaerobic conditions were not characterized, but desmethyl misonidazole (RO-07-9963) and the 2-amino-imidazole derivative of misonidazole (1-[2-aminoimidazol-1-yl]-3-methoxy-2-propanol) were excluded as possible structures.     

Effect of alcohol and aldehyde dehydrogenase inhibitors on the toxicity of 3-nitropropanol in rats

The nitrocompounds 3-nitropropanol (NPOH) and 3-nitropropionic acid (NPA) were shown to be equally toxic when injected intraperitoneally into male Wistar rats. The LD50 for NPOH was 0.58 mmol/kg and for NPA it was 0.56 mmol/kg. NPOH was rapidly metabolized to NPA but this conversion was suppressed by prior administration of ethanol or 4-methylpyrazole to inhibit alcohol dehydrogenase. Administration of ethanol or 4-methylpyrazole before NPOH protected rats from intoxication. However, if the alcohol dehydrogenase inhibitors were given after the nitroalcohol, toxicity still occurred. Administration of the aldehyde dehydrogenase inhibitor diethyldithiocarbamic acid had little effect on the conversion of NPOH to NPA and did not alter the toxicity of NPOH. It was concluded that NPOH and NPA are equally toxic to rats but that NPOH is toxic due to its being rapidly converted to NPA.     

Effects of tolbutamide on insulin binding to isolated fat cells of the rat

In isolated fat cells of the rat the in vitro and in vivo effects of tolbutamide on insulin binding and insulin response were studied. 450 mg tolbutamide/kg/day given for 7 days significantly increased the binding of insulin to isolated adipocytes. The binding curves reflected an increase in the number of receptor sites rather than in the affinity. The effect was associated with an enhanced response to insulin of the adipose tissue, since the fat cells obtained from animals treated with tolbutamide converted significantly more glucose to lipids in the presence of insulin than those obtained from the control group. However, the augmentation of insulin binding sites was observed only at a large tolbutamide dosage, which reduced the pancreatic insulin content, the secretory response of the isolated pancreas, and the serum insulin levels. Smaller doses, sufficient to produce metabolic effects via a stimulation of insulin secretion, did not provide additional insulin binding sites. When added in vitro to the binding assay or to adipose tissue incubated for 16 h, tolbutamide failed to increase insulin binding of the fat cells. It is suggested, therefore, that the effects produced by tolbutamide after in vivo treatment reflect an indirect rather than a direct action of the sulphonylurea.     

Effects of pentagastrin,histamine, carbamylcholine and catecholamines on gastric secretion,motility and emptying in the rat

We developed a simplified method for the simultaneous measurement of gastric secretion and gastric motility in the rat. By using this method, we investigated the actions of pentagastrin, histamine, carbamylcholine and catecholamines on the stomach. Carbamylcholine stimulated acid secretion and induced contraction of the stomach, but norepinephrine tended to inhibit acid secretion and induced relaxation of the stomach. Histamine induced relaxation first and then contraction after 2–5 min. Pentagastrin induced a relaxation that did not show dose dependence, but tachyphylaxis. Isoproterenol, which stimulated acid secretion in our experiments, induced relaxation of the stomach in a dose-dependent manner. We also investigated the relationship between gastric motility and gastric emptying. Carbamylcholine caused an enhancement of gastric emptying, but isoproterenol caused its suppression. Pentagastrin, histamine and norepinephrine did not affect gastric emptying. As shown in the results of our experiments, carbamylcholine caused stimulation of acid secretion, contraction of the stomach, and enhancement of gastric emptying. However, other secretagogues did not always induce contraction of the stomach or increase gastric emptying.