Inhibition of high affinity choline uptake: Structure activity studies

Sodium-dependent, high affinity choline uptake in brain synaptosomes was inhibited by various choline analogues. When substituents were placed on the quaternary nitrogen, the compounds became weaker inhibitors as the bulk of the substituents increased. None of the compounds with changes in the hydroxyl group part, except for a bis-quaternary compound, were potent inhibitors. Increasing the oxygen-nitrogen distance resulted in a weaker inhibition. Data from crystallographic studies [M. E. Senko and M. Templeton, Acta crystallogr. 13, 281 (1960); and F. G. Canepa, Nature, Lond.207, 1152 (1965)] and inspection of Dreiding molecular models suggest an optimal oxygen-nitrogen distance of about 3.3 Å. These results support earlier suggestions that a hydroxyl group and a quaternary nitrogen are necessary for interaction with the carrier.     

Inhibition of iron-stimulated catecholamine degradation by the iron-chelators DETAFAC and Desferal: Potentially useful laboratory agents

The addition of ferrous sulfate to phosphate buffer at pH 7.4 brought about a large increase in the rate of autoxidation of dopamine or norepinephrine. The iron-chelating agents diethylenetri-aminepentaacetic acid (DETAPAC) and desferroxamine (Desferal) inhibited both the baseline as well as the iron-stimulated autoxidation of these catecholamines. Lesser inhibitory effects were observed with another iron-chelating agent, namely EDTA. In other experiments, DETAPAC or Desferal had little effect on the autoxidation of 6-hydroxydopamine in the absence of added ferrous sulfate, but both inhibited the ferrous sulfate-stimulated autoxidation. In contrast, both the baseline and ferrous sulfate-catalyzed rate of oxidation of 6-hydroxydopamine were greatly stimulated by EDTA addition. DETAPAC and Desferal appear to be useful experimental tools that may be added to solutions containing catecholamines to prevent their degradation.     

Inhibition of glycolysis of mammalian cells by misonidazole and other radiosensitizing drugs: Prevention by thiols

Prolonged anaerobic incubation of Ehrlich ascites tumor cells and Chinese hamster V79–379A cells with misonidazole, desmethylmisonidazole, or niridazole led to inhibition of both glucose consumption and lactate formation. This effect was measured in cells washed free of the nitro compounds and resuspended in fresh buffer or medium. The degree of inhibition of glucose utilization was related to drug concentration, and to the rate of metabolic reduction, as measured under aerobic conditions by KCN-insensitive oxygen consumption. Misonidazole-induced inhibition of glycolysis developed concurrently with depletion of intracellular non-protein thiol (NPSH) and was protected against by the presence of cysteamine, cysteine and, to some extent, GSH in the cell incubate. These findings suggest reaction of reduced drug intermediates with thiol-containing enzymes. The glutathione-reactive agent diethyl maleate was used to deplete 90% of the endogenous NPSH, but this depletion did not alter the effects of misonidazole on glycolysis.     

Studies on the arachidonic acid cascade—I: Inhibition of phospholipase A2in vitro and in vivo by several novel series of inhibitor compounds

A series of compounds have been discovered which are potent inhibitors in vitro of hog pancreas, cobra venom, and bee venom phospholipase A₂. Collagen-induced aggregation of human platelets was prevented by representatives of this series. The inhibition was reversed by aggregatory concentrations of arachidonate, indicating the hydrolysis of esterified arachidonate had been prevented. In the isolated perfused guinea pig lung sensitized to ovalbumin, the release of prostanoids, resulting from a challenge dose of the antigen, was prevented by exemplars of these compounds. Subsequent administration of arachidonate in the presence of the inhibitor resulted in full prostanoid synthesis and secretion, again indicating that the block was at the phospholipase level. Administration of some of these compounds to guinea pigs by subcutaneous or intraperitoneal routes delayed the onset, and decreased the severity, of erythema induced in depilitated skin by the controlled application of ultraviolet light. This result was, also, consistent with phospholipase A₂ inhibition. Kinetic experiments with hog pancreas phospholipase A₂ demonstrated that, with representatives of this series, the inhibition induced was noncompetitive, and appropriate dissociation constants have been calculated.     

Inhibition of human elastase from polymorphonuclear leucocytes by gold sodium thiomalate and pentosan polysulfate (SP-54)

Human lysosoma) elastase, the serine proteinase from the azurophil granules of polymorphonuclear leucocytes, is inhibited by gold thiomalate and pentosan polysulfate (SP-54®). The kinetic mechanism of the inhibition was studied using succinyl-alanyl-alanyl-prolyl-valyl-4-methyl-7-coumarylamide and (t-butyloxycarbonyl-alanyl-p-nitrophenylester as subsrates. The degree of inhibition was also tested using insoluble elastin as substrate. Independent of the substrate, the maximal inhibition of elastase by gold thiomalate and pentosan polysulfate was 40% and 60%, respectively. Pentosan polysulfate behaved as a simple intersecting, hyperbolic, non-competitive inhibitor and k′_i. the dissociation constant of the E-S-I complex, was 1.8 × 10^?7M. The interaction between inhibitor and enzyme is driven by electrostatic forces. Gold thiomalate showed a hyperbolic mixed type inhibition (intersecting, slope-hyperbolic, intercept-hyperbolic, non-competitive inhibition) with k′_i = 5.4 × 10^?5M.The overall kinetic mechanism of lysosomal elastase conforms to that of other serine proteinases. With both the ester and peptide substrates the rate limiting step of the reaction has been identified with the formation of the acyl-enzyme.     

Inhibition of dog kidney Na+, K+-ATPase activity by procaine, tetracaine and dibucaine

The potency of local anesthetics as inhibitors of Na+, K+-ATPase and K+- NPPase activities correlated with lipid solubility. The order of potencies was: dibucaine greater than tetracaine much greater than procaine. Na+-ATPase activity was remarkably more sensitive to inhibition by tetracaine and procaine, and inhibitory potency did not correlate with lipid solubility. The order of potencies for inhibition of Na+-ATPase activity was: tetracaine greater than dibucaine greater than procaine. We examined interactions between the local anesthetics and monovalent cations in an attempt to explain this observation. Inhibition of Na+-K+-ATPase by tetracaine and dibucaine was competitive with respect to Na+, and inhibition of Na+-ATPase activity by all three agents was competitive with respect to Na+. Inhibition of Na+, K+-ATPase activity by procaine and tetracaine was competitive with respect to K+, and inhibition of K+- NPPase activity by all three agents was competitive with respect to K+. Dibucaine, the most lipid soluble agent, was equipotent as an inhibitor of all three activities and was generally less effective as a competitor with respect to activation by monovalent cations. These results suggest that dibucaine may interact nonspecifically with membrane lipids to inhibit enzyme activity whereas less lipid soluble agents, such as tetracaine and procaine, may interact more selectively with cation binding sites. It appears that the presence of K+ in the assay medium specifically decreases the inhibitory potency of tetracaine and procaine. Direct competition between these agents and K+ may prevent inhibition or, alternately, the presence of K+ may convert the enzyme to a conformation less susceptible to inhibition by agents of low to intermediate lipid solubility.     

Inhibition of prolyl hydroxylase activity and collagen biosynthesis by the anthraquinone glycoside, P-1894B, an inhibitor produced by Streptomyces albogriseolus

P-1894B, a potent prolyl hydroxylase inhibitor produced by Streptomyces albogriseolus subsp. No. 1894, inhibited about 50% of the activity of purified chick embryo prolyl hydroxylase at a concentration of 2.2 x 10(-6) M. The inhibition was noncompetitive with respect to (Pro-Pro-Gly)5 with a Ki of 1.8 x 10(-6)M. When excess amounts of ferrous ions or ascorbate were added to the reaction mixture, the inhibition was slightly reversed. P-1894B at a dose of 0.15 mg/kg reduced the hydroxylation of peptidyl proline and caused a significant inhibition of collagen biosynthesis in the uterus of the immature rat stimulated by the administration of estradiol-17 beta.     

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.     

Heme biosynthesis in the heart

The rates of biosynthesis of heme a and heme b in hearts of fed and fasted rats were measured using an isolated heart perfusion system. δ-Aminolevulinic acid synthetase activity was decreased in hearts of fasted rats to about 30% of values in hearts obtained from fed rats. [¹⁴C]Glycine incorporation into hemes a and b of cardiac tissue obtained from fasted rats was also decreased to about 30% of values obtained in hearts from fed rats. Cobalt addition to the perfusion fluid led to a decrease in cardiac δ-aminolevulinic acid synthetase activity just as cobalt administration to rats does in vivo. These studies strongly suggest that δ-aminolevulinic acid synthetase activity regulates the rate of synthesis of hemes a and b in the heart.     

The swift increase in alcohol metabolism: Inhibition by propylthiouracil

Administration of a single large dose of ethanol (5 g/kg) to rats elevates the rates of ethanol metabolism and of oxygen consumption in perfused livers in 2–3 hr. Pretreatment with the antithyroid drug propylthiouracil (PTU) for 10 days abolished both of these effects. Under all treatment conditions studied (controls; PTU-pretreatment; acute ethanol treatment; PTU-pretreated + acute ethanol treatment), a significant correlation between ethanol metabolism and oxygen consumption was observed (r = 0.86). It is concluded that a normal thyroidal state is required to evoke the swift increase in alcohol metabolism (SIAM) and an elevation of oxygen consumption.     

Regulation of NADPH-dependent mixed-function oxidation in perfused livers: Comparative studies with sorbitol and ethanol

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD⁺/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP⁺/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.     

Nitroheterocycle metabolism in mammalian cells. Stimulation of the hexose monophosphate shunt

Misonidazole, SR-2508, nitrofurazone and other nitroheterocycles stimulated release of 14CO2 from [1-14C]glucose but not from [6-14C]glucose when incubated with mouse Ehrlich ascites cells or human A549 lung carcinoma cells in vitro. This demonstrated that the nitro compounds activated the hexose monophosphate shunt and is evidence that an important pathway of nitro reduction in these cell lines is electron transfer from NADPH-dependent cytochrome c reductase to the nitro group. Shunt activity was stimulated under both aerobic and anaerobic conditions. For catalase-free Ehrlich cells, aerobic effects were greater than anaerobic, indicating that NADPH was used for reduction of H2O2, via GSH peroxidase and reductase, as well as for one-electron nitro reduction, under aerobic conditions. Several of the compounds tested stimulated 14CO2 release from [2-14C]glucose as well as from [1-14C]-glucose. This shows that the cellular requirement for NADPH, in the presence of nitro drug, was great enough to cause recycling of pentose phosphates. Recycling could decrease the availability of ribose-5-P needed for nucleic acid synthesis, which could partly explain the inhibition of DNA synthesis observed upon prolonged aerobic incubation of cells with nitro compounds. Comparison of the rate of disappearance of nitrofurazone from anaerobic A549 cell suspensions with the rate of 14CO2 release suggests that the drug reduction in this cell line was catalyzed almost entirely by NADPH-requiring enzymes.     

Regulation of transmitter synthesis and release in mesolimbic dopaminergic nerve terminals: Effect of ethanol

Slices from rat olfactory tubercle were incubated in freshly oxygenated Krebs-Ringer phosphate (KRP) and in the presence of l-tyrosine [¹⁴C-U] as dopamine (DA) precursor. Thereafter, the newly synthesized [¹⁴C]DA and the [¹⁴C]DA released into the incubation media were isolated by Alumina column, and ion-exchange, chromatography. The presence of K⁺ depolarizing concentrations (25–70 mM) in the incubation media markedly increased the formation of [¹⁴C]DA from [¹⁴C]tyrosine, following a rather complex and biphasic pattern. Dibutyryl cyclic AMP (dB-cAMP) and theophylline also increased the formation of newly synthesized [¹⁴C]DA. Ethanol (0.2 to 0.4%. w/v significantly blocked the stimulation of [¹⁴C]DA biosynthesis that was induced by low K⁺ depolarizing concentrations (25 mM) and by dB-cAMP (5 × 10^?4M) or theophylline (1 × 10³M). In contrast, only higher ethanol concentrations (0.8 to 1.1%, w/v) blocked the increase in DA formation induced by high K⁺ depolarizing concentrations (40 and 55 mM). Potassium depolarization (40 mM) markedly evoked the release of newly synthesized [³H]DA or [³H]DA previously taken up by the slices. The release was shown to be dependent upon the presence of Ca²⁺ and inhibited by an excess of Mg²⁺ (12mM). Ethanol (0.8 to 1.1%, w/v) produced no effect on K⁺-induced release of [³H]DA. The model described in this paper can be used as a simple experimental tool to study neurotransmitter synthesis and release from nerve terminals belonging to the mesolimbic dopaminergic system. The results reported suggest the existence of at least two mechanisms by which neuronal depolarization increases transmitter formation in mesolimbic dopaminergic terminals. Ethanol, at relatively low concentrations, seems to produce a specific inhibitory effect upon the mechanism that predominates under low depolarizing conditions. The possibility is raised that the effects described for ethanol may play a role in the neuropharmacological responses induced by this agent in vivo.     

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.