Tight-binding inhibitors—IV. Inhibition of adenosine deaminases by various inhibitors

Three ADA (adenosine deaminase) inhibitors, DHMPR (1,6-dihydro-6-hydroxymethyl purine ribonucleoside); EHNA [erythro-9-(2-hydroxy-3 nonyl)adenine] ; and deoxycoformycin [(R)-3-(2-deoxy-β-d-erythro-pento-furanosyl)-3, 6,7,8-tetrahydroimidazo[4,5-d] [1,3-diazepin-8-ol] or Covidarabin, were compared with regard to their inhibitory behavior with ADAs from human erythrocytes and calf intestine. Marked differences in the times required for establishment of steady state between the enzyme and inhibitors were observed, e.g. DHMPR, virtually instantaneous; EHNA, 2–3 min; and deoxycoformycin, many hr. The parameters of the inhibition of human erythrocytic ADA by deoxycoformycin were as follows: the association rate constant (k₁) = 2.6 × 10⁶ M^?1 sec^?1 ; the dissociation rate constant of the enzyme-inhibitor complex (k₂) = 6.6 × 10^?6 sec^?1; K_i (from $\text{k}{}_2\text{k}{}_1\text{) = 2.5 × 10}{}^{\mathrm{?12}}\text{M}$ and K_i (from I₅₀) = 1.5 × 10^?11 M. The K_i values for EHNA and DHMPR, as determined by classical methods after attainment of steady state, were 1.6 × 10^?9 and 1.3 × 10^?6 M, respectively, for human erythrocytic ADA. The kinetic parameters for EHNA and calf intestinal ADA were as follows: K_i = 6.5 × 10^?9 M (by the method of I₅₀); k₁ = 0.7 × 10⁶ M^?1 sec^?1' and k₂ = 4.6 × 10^?3 sec^?1. On the basis of K_i values, the inhibitors. DHMPR, EHNA and deoxycoformycin (a transition state analog), were classified as readily reversible, semi-tight-binding and tight-binding inhibitors. The difficulties encountered in the kinetic analyses of different types of inhibitors and the methods for dealing with the problems of these inhibitors are discussed.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Tight binding inhibitors--VIII. Studies of the interactions of 2'-deoxycoformycin and transport inhibitors with the erythrocytic nucleoside transport system

Studies were performed to extend earlier observations that the rate-limiting step in the inactivation of intraerythrocytic human adenosine deaminase (ADA) by 2'-deoxycoiormycin (dCF) is the nucleoside transport system (NTS). The NTS inhibitors 2-amino-6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-D-ribofuranosyl purine (HNBTGR), 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine (NBMPR), 2-amino-6-[(4-nitrobenzyl)Seleno]-9-β-D-ribofuranosyl purine (NBSeGR), dipyridamole and the competitive permeant, uridine, all decreased the rate of ADA inactivation by dCF in a concentrationdependent manner. Lineweaver-Burk plots of $\text{1}\text{k}{}_{\mathrm{\gamma }}$ (where k_γ is the pseudo first-order rate constant for the inactivation of ADA) 1/dCF concentrations were linear, giving a K_m tor dCF tor the NTS of 6 × 10^?7 M. The maximal k_γ calculated by extrapolation to infinite dCF concentrations was 6 × 10^?3 per sec which corresponds to a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 115 sec. Similar plots for experiments with the NTS inhibitors and uridine yielded classic patterns of competitive inhibition for NBMPR, HNBTGR, NBSeGR and uridine, whereas with dipyridamole a pattern of non-competitive inhibition was obtained. Dissociation or inhibition constants have been reported for several of these compounds (determined by other methods) and values similar to these were obtained. Inhibition by dipyridamole was non-competitive (k_l = 2.5 × 10^?7 M) and was of a bi-phasic nature with respect to time. Dipyridamole caused rapid and irreversible inhibition for the first 7–15 min with slow and progressive but reversible inhibition thereafter. These observations are consistent with the hypothesis that NBMPR, HNBTGR, NBSeGR and uridine interact with the same site on a macromolecular component of the NTS that forms ligands with dCF. The behavior of dipyridamole appears more complex and will require more extensive study.     

The effect of bencyclane on the oxidative phosphorylation in isolated mitochondria of heart and liver

Our experiments were designed to localize the inhibitory influence of bencyclane² on the process of oxidative phosphorylation in isolated heart and liver mitochondria. The following results were obtained: (1) The state-3-respiration of rat liver and rabbit heart mitochondria was inhibited by bencyclane. This inhibition was dependent on the substrate used as energy donator, being much more pronounced with glutamate $\text{(}\text{ed}{}_{50}\text{= 3.17 × 10}{}^{\mathrm{?8}}\text{or}\text{1.85 × 10}{}^{\mathrm{?7}}\text{moles/mg}$ of protein, respectively) than with succinate $\text{(}\text{ed}{}_{50}\text{= 3.4 × 10}{}^{\mathrm{?7}}\text{or}\text{4.78 × 10}{}^{\mathrm{?7}}\text{moles/mg}$ of protein, respectively). Since the 2,4-dinitrophenol stimulated respiration was equally inhibited, and glutamate transfer through the mitochondrial membrane not influenced, we assume the NADH-coenzyme-Q-reductase to be the site of interaction at the molecular level. (2) Bencyclane stimulates the state-4-respiration of isolated mitochondria with $\text{concentrations}\text{\$}\text{?}\text{= 10}{}^{\mathrm{?5}}\text{M}$. This effect depends on the molar bencyclane concentration of the incubation medium, and is not abolished by the addition of atractyloside, oligomycin or ruthenium red. Therefore, it is suggested that uncoupling of oxidative phosphorylation is the reason for this bencyclane effect. Theoretically, both of the described effects result in a reduction of the amount of ATP in the living cell. Possible consequences on myocardial function and the cardiovascular system are discussed in terms of previously published data in this field.     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Concentrations of nitrate in normal human urine and the effect of nitrate ingestion

A method suitable for the analysis of nitrate in human urine was developed. Normal urinary concentrations of nitrate in urine of human volunteers in Dade County, Florida, where the drinking water contains negligible amounts of nitrate, averaged 47.6 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$ (SD = 17.3). On a vegetable and preserved-meat-free diet, the nitrate concentration was reduced (10 to 30 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$), but, on nitrate-supplemented drinking water, the urinary concentration rose to a range of 34–87 ppm of $\text{NO}{}_3{}^{\mathrm{?}}$. A high vegetable diet resulted in peak urinary nitrate concentrations of 270–425 ppm. These results indicated that nitrate in drinking water is a factor in determining urinary nitrate concentration, but that vegetable ingestion is of greater significance.     

The effect of halothane on the stability of Ca2+ transport activity of isolated fragmented sarcoplasmic reticulum

The effects of the inhalation anaesthetic agent, halothane (CF₃CHBrCl), on the stability of the calcium transport system of isolated rabbit white skeletal muscle sarcoplasmic reticulum have been studied. Calcium transport activity was unaffected when suspensions of sarcoplasmic reticulum vesicles were preincubated at 37° and pH 6.8 at concentrations of halothane below 5 mM, but was progressively inactivated at higher concentrations. (Ca²⁺,Mg²⁺)-ATPase activity was enhanced during inactivation of calcium transport. At pH 6.3 and 5.8, halothane increased the first order rate constants of inactivation and effects were noted in the anaesthetic range of concentration (1–2 mM). The inulin inaccessible space of membrane vesicles did not change appreciably during the period of treatment with halothane, excluding increased permeability as an explanation of the inhibition of calcium accumulation. Inactivation was irreversible and highly temperature dependent, with an activation energy of 52.7 kcal/mol. Calcium ions had a protective effect against inactivation (K_{0.5 (Ca2+) = 1.5 × 10^?6M}), as did ATP ($\text{K}{}_{\mathrm{<mtext>0.5\; (</mtext><mtext>Atp</mtext><mtext>)</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$). It is concluded that mild acid conditions and halothane act synergistically during inactivation of the calcium transport system of sarcoplasmic reticulum membranes. These studies suggest that halothane interacts with the (Ca²⁺, Mg²⁺)-ATPase protein at the ATP-specific binding site or that it disrupts protein-lipid associations in the membrane. In either case the destabilizing effect of halothane may be modified by the conformational state of the protein.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Dimethyl sulfoxide: Inhibition of acetylcholinesterase in the mammalian heart

The heart rate of the isolated, perfused, working rat heart was significantly and equally depressed by 1 × 10^?6 M acetylcholine (ACh) and by 6 × 10^?5 M 4-ketoamyltrimethylammonium (4K), a cholinomimetic agonist. Dimethyl sulfoxide (DMSO) (10 μl/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 μl/ml, 140 mM final concentration) alone had no significant effect upon heart rate when added to the perfusate in incremental additions of 1 μl · (ml perfusate)^?1 · min^?1 over a 10-min period. The specific activity of atrial homogenate cholinesterase was $\text{48.8 ± 3.46}\text{nmoles}\text{·}\text{min}{}^{\mathrm{?1}}\text{· (}\text{mg protein}\text{)}{}^{\mathrm{?1}}\text{(}\text{mean}\text{±}\text{S.E.M.}\text{), 38.2 ± 1.60}$ for butyrylcholinesterase, and 11.2 ± 0.86 for acetylcholinesterase (AChE). True AChE activity (measured in the presence of a maximally effective concentration of tetraisopropylpyrophosphoramide) had a V_max of 13.4 ± 0.17 nmoles · min^?1 · (mg protein)^?1 and an apparent K_m value of 1 × 10^?4 M acetylthiocholine. At this K_m substrate concentration, DMSO inhibited atrial AChE activity (I₅₀ = 9 μl/ml). At the concentration tested, DMSO inhibited atrial AChE and potentiated ACh effects.     

Dopaminergic effects of buspirone,a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methyxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [³H]spiroperidol from striatal binding sites, with an ic₅₀ (1.8 × 10^?7 M), comparable to that of clozapine ($\text{ic}{}_{50}\text{= 1.4 × 10}{}^{\mathrm{?7}}\text{M}$) but considerably lower than that of haloperidol (4.7 × 10^?9 M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Correlation of biologic data with physico-chemical properties among the vinca alkaloids and their congeners.

Displacement of [³H]vinblastine binding to tubulin by other Vinca alkaloid derivatives has been demonstrated to be a competitive process, allowing for determination of the association constant of each drug. Correlation of LD₅₀ data and anti-P-388 activity was found with log P and log K_a, according to the equations: $\text{log}\text{LD}{}_{50}\text{= 0.129 (}\text{log}\text{P)}{}^2\text{? 0.522}\text{log}\text{P ? 0.479}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 4.652}\text{log}\text{P ? 388 = 0.222 (}\text{log}\text{P)}{}^2\text{? 1.059}\text{log}\text{P ? 0.520}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 5.366}$. Vincristine and desacetylvinblastine were the two most active agents in this series. That the latter drug had significant biologic activity was of considerable interest, since it is known to be a human metabolite.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

The role of lipid,free radical initiator,and oxygen on the kinetics of lipid peroxidation

The relationship between the concentration of unsaturated lipid, free radical initiator, and oxygen concentration on the kinetics of lipid peroxidation was determined. The rate of lipid peroxidation was studied with the thiobarbituric acid (TBA), diene conjugation (DC), and ferrithiocyanate (Fe-SCN) methods. The rate of peroxidation was half-order with respect to unsaturated lipid, initiator, and oxygen. The half-order relationship could be expressed as: $\text{rate}\text{= (}\text{fk}{}_1\text{k}{}_2\text{k}{}_3\text{k}{}_6{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{azobisisobutyronitrile}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$$\text{(}\text{RH}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{O}{}_2\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. The half-order relationship was found with linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids. A linear relationship existed between the logarithm of unsaturation and the rate of peroxidation. No peroxidation of linolenic acid was indicated when the DC method was employed, but was when the TBA and Fe-SCN methods were used.