Cholinergic elements in a human choriocarcinoma cell line

Components of the cholinergic system have been identified in the JEG choriocarcinoma cell line. [³H]Quinuclidinyl benzilate (QNB) was used to identify high affinity muscarinic binding sites in a whole cell preparation. Specific binding was saturable with respect to QNB concentration and revealed a binding site density of 27 fmoles/mg protein. The bimolecular rates of association, 2.24 × 10⁷ M^?1 min^?1, and dissociation, 4.2 × 10^?3min^?1, revealed a dissociation constant (K_d) of 180 pM which agreed closely with that derived from saturation isotherms, 245 pM. Muscarinic antagonists and agonists were able to compete effectively for these binding sites, whereas non-muscarinic compounds were not. Cholinesterase activity was also demonstrated with substrate preference consistent with that of acetyl-cholinesterase (acetylcholine > acetyl-β-methylcholine > butyrylcholine) hydrolyzing 2.42 ± 0.19 × 10^?3μmoles acetylcholine·min^?1 ·(mg protein)^?1. No choline acetyltransferase activity was detected in these cells, however.     

Depression of myocardial contractility in acute iron toxicity in rabbits

The hemodynamic effects of acute iron toxicity were evaluated in rabbits. A lethal dose of iron (200 mg/kg as ferrous sulfate) was administered into the duodenum of seven rabbits as heart rate, right atrial pressure, arterial pressure, cardiac output, left ventricular pressure, right ventricular developed force, and arterial pH were monitored. Hemodynamic variables were compared to those in a control group of animals (n = 7; no iron) and to those in a group of animals (n = 7) receiving the same dose of iron but with the addition of an iv infusion of sodium bicarbonate to prevent iron-induced acidosis. Our results demonstrate that an acute lethal dose of iron in the rabbit: (1) increases systemic vascular resistance, (2) does not alter filling pressure of either the right or left ventricle, and (3) depresses stroke volume, cardiac output, right ventricular force, and left ventricular $\text{dP}\text{dt}$. Prevention of acidosis does not prevent the hemodynamic alterations induced by the administration of iron. We conclude that acute iron toxicity significantly depresses myocardial contractility. Diminished myocardial contractility appears to be important in the pathogenesis of iron-induced shock in the rabbit.     

Plasma protein binding and interaction studies with diflunisal,a new salicylate analgesic

The binding of diflunisal to human serum albumin and normal human plasma has been studied by equilibrium dialysis at 37°, pH 4. The plasma protein binding data were analysed according to a Scatchard model with two independent classes of binding sites. The number of binding sites and the corresponding association constants have been estimated by nonlinear least-squares regression analysis: N₁ = 2.1, k₁ = 5.28 × 10⁵M^?1, N₂ = 7.7 and K₂ = 0.17 × 10⁵M^?1. At a difluni concentration of 50 μg/ml on average 99.83 per cent of the drug was bound to plasma proteins. The in vitro plasma protein binding of diflunisal was impaired by salicylic acid and phenprocoumon, while diflunisal itself was displaced from its primary binding sites in plasma by salicylic acid and bilirubin. Tolbutamide had no effect on the binding of diflunisal to plasma proteins.     

Role of zinc as an activator of mitochondrial function in rat liver

The effects of zinc on the enzymes of hepatic mitochondria were investigated in rats that had been given zinc sulfate (10 mg Zn2+/100 g body wt) p.o. Administration of zinc caused a marked elevation of succinate dehydrogenase, glutamate dehydrogenase, cytochrome c oxidase and ATPase activities, whereas it did not cause significant changes in pyruvate carboxylase, malate dehydrogenase and isocitrate dehydrogenase activities. The effect of zinc as a function of time was greatest on succinate dehydrogenase. Zinc also produced a marked elevation of ATP concentration in the hepatic cytosol and a corresponding increase in ATPase activity in the hepatic mitochondria. Zinc content of the inner membrane of mitochondria was raised significantly by administration of zinc. The removal of zinc by washing in 10 mM EDTA caused a significant decrease of the increased succinate dehydrogenase activity caused by administration of zinc, while it did not lower ATPase activity. The addition of zinc in amounts of 10-10(3) ng Zn2+ per mg protein produced a significant increase in succinate dehydrogenase activity in the inner membrane of mitochondria, whereas ATPase activity was elevated significantly at 10(3)-10(4) ng Zn2+ per mg protein, indicating that zinc activated succinate dehydrogenase more sensitively than ATPase. The present investigation suggests that zinc taken up by hepatic mitochondria stimulates the electron transport system and oxidative phosphorylation and, as a result, increases the ATP concentration in the hepatic cytosol.     

Effect of acute carbon monoxide exposure on cardiopulmonary function of the awake rat

Unanesthetized male Sprague-Dawley rats were exposed for 20 min to 0 (control), 2000, or 4000 ppm carbon monoxide, and cardiopulmonary responses were evaluated. Venous blood samples were taken prior to exposure and at 2, 4, 7, 11, 15, and 20 min during exposure. Responses for CO-exposed animals were compared at 20, 30, 40, 50, and 60% carboxyhemoglobin (COHb), and these values were compared to control values at similar times during exposure. Fifty percent COHb was attained within 4 to 8.5 min at 4000 ppm and 11 to 14 min at 2000 ppm. For both CO exposure groups, mean arterial pressure first decreased at 40% COHb and fell to 69% of baseline at 60% COHb. Tidal volume and breathing frequency increases resulted in minute volume elevations of 52 (2000 ppm) and 77% (4000 ppm) at 60% COHb. Minute volume was elevated at 50 and 60% COHb at 4000 ppm, but was not elevated until 60% COHb for 2000-ppm exposures. The earlier ventilatory stimulation at 4000 ppm may have been a by-product of increased agitation observed in rats exposed to the higher concentration.     

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.     

Conversion of N-hydroxyamphetamine to phenylacetone oxime by rat liver microsomes

These in vitro studies indicate that N-oxidation of N-hydroxyamphetamine (NOHA) by rat liver homogenates yields phenylacetone oxime (PAOx) as the major metabolite. This oxidation was NADPH and oxygen dependent but was not appreciably increased in microsomes from phenobarbital-pretreated animals. The addition to microsomal incubations of Superoxide dismutase (SOD), catalase (CAT), azide or mannitol did not alter the rate of oxidation, suggesting that O₂su?. H₂O₂, or OH^. are not involved in this process. The reaction was minimally inhibited by a 2:1 ratio of CO/O₂, and there was no significant reduction in the formation of product by the presence of diethylaminoethyl diphenylvalerate (SKF-525A) or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) in micromolar concentrations. Thus, although this NADPH-dependent N-oxidation pathway was catalyzed by rat hepatic microsomes, the data suggest that it was not a cytochrome P-450 mediated monooxygenase reaction.     

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.     

Disposition of rubratoxin B in the rat

Excretion, tissue concentrations in the kidney and liver, and pharmacokinetic parameters estimated from plasma blood concentrations were determined for rats given a single ip dose of [¹⁴C]rubratoxin B (0.05 mg dissolved in propylene glycol). By 7 days, 80% of the administered radioactivity had been excreted into the urine (41.7%) and feces (38.7%). Urinary excretion was primarily as the parent compound, accounting for 75% of the radioactivity excreted by 7 days. Elimination of radio-activity from the kidneys was monophasic with a half-life of 97.35 hr. Elimination of radioactivity from the liver was biphasic, with a half-life of 13.66 hr for the slow phase. Elimination of rubratoxin B and [¹⁴C]rubratoxin B-derived radioactivity (radioactivity derived from both the parent compound and metabolites) from the plasma was biphasic. The rapid phases of elimination had half-lives of 2.57 and 1.08 hr, and the slow phases had half-lives of 60.80 and 100.46 hr for rubratoxin B and [¹⁴C]rubratoxin B-derived radio-activity respectively. The long plasma half-life of rubratoxin B is suggestive of enterohepatic circulation. The concentration of radioactivity was greatest at 1 hr in the liver and 2 hr in the plasma. Except for the first few hours following injection, the concentration of radioactivity in the liver never exceeded significantly that in the plasma, suggesting a passive absorption process. No glucuronide or sulfate conjugates were detected in the plasma or urine.     

Impaired drug metabolism in experimental cirrhosis in the rat

Cirrhosis was produced in the rat by chronic administration of phenobarbital and carbon tetrachloride for 10 weeks. The metabolism of three substrates, aminopyrine. hexobarbital and propranolol, has been investigated in a 9000 g supernatant fraction of liver homogenate and in intact hepatocytes in cirrhotic livers and compared to appropriate phenobarbital-treated controls. In the 9000 g supernatant preparation, the maximal velocity of metabolism for each substrate and the cytochrome P450 concentration were reduced significantly in cirrhotic livers, while total protein and DNA concentration remained unchanged. In intact hepatocytes, the V_maxfor each substrate was reduced, while the apparent K_m remained unchanged. In both in vitro systems, the metabolism of propranolol and of hexobarbital was influenced by cirrhosis to a greater extent than that of aminopyrine. It is concluded that the carbon tetrachloride-phenobarbital model of cirrhosis in the rat is a suitable model in which to study the effects of chronic liver disease on drug disposition.     

Mechanism for potentiation of warfarin by phenylbutazone. Inhibition of vitamin K-dependent carboxylation and prothrombin synthesis by phenylbutazone in preparations from rat liver

Phenylbutazone potentiated the anticoagulant effects of racemic warfarin and of the individual enantiomers to similar extents in the rat. This indicates that the phenylbutazone did not act stereospecifically on the enantiomers, as it does in humans. Phenylbutazone doubled the turnover rate of warfarin in plasma, but it did not increase the amount of the anticoagulant in liver or the amount excreted in urine. The drug had no effect on plasma disappearance of [3H] or on hepatic levels of [3H] vitamin K1 or of its chief metabolite, [3H] vitamin K1 epoxide, after injection of [3H] vitamin K1. Phenylbutazone, however, at concentrations of 0.5 to 2.8 mM inhibited vitamin K-dependent carboxylation of a synthetic pentapeptide substrate in liver microsomes by 40-88 per cent. Vitamin K-dependent protein carboxylation was also inhibited by about 40 per cent in microsomes and post-mitochondrial supernatant fluid at drug concentrations of 2.8 to 4.8 mM. Most importantly, prothrombin synthesis was inhibited in post-mitochondrial supernatant fractions by 19 and 39 per cent at drug concentrations of 2.8 and 4.8 mM respectively. The inhibition of both carboxylation and prothrombin synthesis appears to have been of sufficient magnitude to account for the potentiation by phenylbutazone observed in vivo. The calculated hepatic level of phenylbutazone during potentiation was around 3 mM, a concentration that produced inhibition in vitro.     

Enzyme inhibition and the toxic action of moniliformin and other vinylogous α-ketoacids

The inhibition of two thiamine-requiring enzymes by the potent mycotoxin, moniliformin (1-hydroxycyclobutene-3,4-dione), was investigated. Rat brain transketolase and pyruvate dehydrogenase were inhibited 25 percent by 10^?9 M moniliformin. Studies carried out to determine if moniliformin causes enzyme inhibition by reaction with thiamine were negative. Varying the hydroxycyclobutenedione structure by substitution or ring expansion resulted in loss of toxicity and inhibition.     

Mechanism of the protective action of thiol compounds in ethanol-induced liver injury

The protective action of cysteine or mercaptopropionylglycine (MPG) in acute ethanol-induced liver injury has been investigated in the rat. Cysteine accelerated clearance of ethanol and acetaldehyde from blood and liver and prevented an increase in hepatic content of triglyceride and serum ornithine carbamoyl transferase activity. MPG accelerated clearance of ethanol and acetaldehyde less efficiently but prevented an increase in these variables to the same degree. The mode of action of thiol compounds in acute ethanol-induced liver injury has been discussed.     

Effect of 3-aminopicolinic acid on renal ammoniagenesis in the rat

The effects of 3-aminopicolinate, a known activator of phosphoenolpyruvate carboxykinase (PEPCK), on renal ammoniagenesis were studied in normal rats and in rats allowed to recover for 2 days from ammonium chloride induced metabolic acidosis. The administration of 3-aminopicolinate (5 mg/100 g body wt) did not affect glomerular filtration rate, renal blood flow, arterial blood pH, pO₂ or pCO₂ in either normal or “recovered” rats. A significant increase in renal glutamine extraction and total ammonia production was observed in recovered rats, but not in normal rats, after 3-aminopicolinate treatment. Although this compound activated partially purified renal PEPCK, the profile of metabolites from freeze-clamped kidneys was not consistent with an activation of the enzyme. Thus, the enhancement of renal ammoniagenesis in vivo by 3-aminopicolinate was probably due to an effect other than activation of PEPCK.     

Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K_m were roughly similar for both pathways, V_max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ-N-oxidation by the rat lung. Hg²⁺ and Mg²⁺ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.