Effects of catecholamine releasing agents on synaptosomal dopamine biosynthesis: Multiple pools of dopamine or multiple forms of tyrosine hydroxylase?

The effects of agents, which are known to induce release of catecholamines from synaptosomes, were assessed on the synthesis of dopamine from tyrosine, as reflected in the evolution of ¹⁴CO₂ from L-[1-¹⁴C]-tyrosine, in intact rat striatal synaptosomes. At a time when release had occured, whereas reserpine inhibited the synthesis of dopamine from tyrosine, with an ED₅₀ of $\text{1 × 10}{}^{\mathrm{?8}}\text{M}$, tyramine (ED₅₀ of $\text{1 × 10}{}^{\mathrm{?5}}\text{M}$) and (+)-amphetamine (ED₅₀ of $\text{1·4 × 10}{}^{\mathrm{?6}}\text{M}$) enhanced the rate of synthesis. The presence of nialamide (10^?4M) or pargyline (10^?3M) had no effect on synaptosomal dopamine synthesis in the absence or presence of amphetamine, tyramine, or reserpine. Neither reserpine, tyramine, nor amphetamine effected the activity of tyrosine hydroxylase or DOPA decarboxylase in the absence of synaptosomal structural integrity. Nor did these drugs effect the accumulation of [³H]-tyrosine into synaptosomes. The data are consistent with the existence of at least two pools of synaptosomal dopamine, one of which can interact with tyrosine hydroxylase. Two hours after pretreatment of rats with 5 mg/kg (+)-amphetamine, the level of synaptosomal dopamine biosynthesis was decreased by 39%. The rate of dopamine synthesis in synaptosomes from amphetamine-pretreated rats was assessed in the presence of reserpine and tyramine. The data are not consistent with alterations in pool size being the only mechanism affecting synaptosomal dopamine synthesis. A mechanism is discussed involving an equilibrium of tyrosine hydroxylase between active and inactive conformers in the presence of an inhibitory pool of dopamine.     

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.     

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.     

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.     

Effects of haloperidol and apomorphine on catecholamine metabolism in brain slices: Reserpine-like effects of haloperidol

The accumulation, release and catabolism of [³H]dopamine (DA) and [³H]norepinephrine (NE) synthesized from [³H]tyrosine were measured in mouse striatal and substantia nigral slices. Apomorphine inhibited both [³H]NE and [³H]DA accumulation $\text{(}\text{ic}{}_{50}\text{< 10}{}^{\mathrm{?6}}\text{M}\text{)}$, presumably by acting on a presynaptic receptor. Haloperidol (10^?8 M) caused a small, but significant increase in [³H]DA accumulation from [³H]tyrosine in the presence of 26 mM K⁺, possibly reflecting blockade of presynaptic receptors activated by released DA. However, at higher concentrations (10^?6 to 10^?5 M), haloperidol inhibited [³H]DA and [³H]NE accumulation. Reserpine also potently inhibited catecholamine synthesis; chlorpromazine had only a weak effect, and fluphenazine was ineffective. Both haloperidol (10^?5 M) and reserpine (10^?7 M), but not chlorpromazine and fluphenazine, markedly increased the formation of labeled dihydroxyphenylacetic acid (DOPAC) and increased the spontaneous release of labeled DA from striatal slices preloaded with [³H]tyrosine or [¹⁴C]DA. These data suggest that haloperidol has some direct effects on DA metabolism that are unrelated to DA-receptor blockade. Because the effects of haloperidol are apparently independent of DA release, haloperidol may elevate cytoplasmic DA by altering its vesicular storage. This would, in turn, increase the spontaneous release of labeled DA by diffusion, the oxidation of DA to DOPAC by monoamine oxidase, and the end-product inhibition of tyrosine hydroxylase.     

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.     

N-[2(o-iodophenoxy)ethyl]cyclopropylamine hydrochloride (LY121768), a potent and selective irreversible inhibitor of type a monoamine oxidase

The effects of N-[2-(o-iodophenoxy)ethyl]cyclopropylamine hydrochloride (LY121768) on types A and B monoamine oxidase (MAO) assayed with radiocarbon-labeled serotonin and phenyl-ethylamine, respectively, were studied in vitro and in vivo. Type A MAO from rat brain was inhibited in vitro by LY121768 with an ic₅₀ of 4 × 10^?10 M, whereas 2500 times higher concentrations of LY121768 $\text{(}\text{ic}{}_{50}\text{= 1 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ were required to inhibit type B MAO. The inhibition of type A MAO increased with time of incubation of LY121768 with enzyme prior to substrate addition and persisted after dialysis, indicative of irreversible inhibition. The irreversible inactivation was prevented by harmaline, a reversible, competitive inhibitor of type A MAO, indicating a requirement for catalytic activity of MAO in the time-dependent inactivation by LY121768. In rats, LY121768 selectively inhibited type A MAO in brain and in liver at low doses. The inhibition of type A MAO persisted for several days after a single 10 mg/kg i.p. dose of LY121768 and was associated with a significant increase in brain dopamine, norepinephrine and epinephrine concentrations and a significant decrease in the concentration of the dopamine metabolites, homovanillic acid and 3.4-dihydroxyphenylacetic acid. The inactivation of type A MAO by LY121768 in vivo was prevented by co-administration of harmaline, indicating a similar mechanism for the in vivo inactivation as for the in vitro inactivation of MAO by LY121768. A reasonable inference from these data and from previous literature is that LY121768 acts as a “suicide substrate” for MAO and inactivates the enzyme by formation of a reactive intermediate which binds covalently to the enzyme. The presence of iodine in the LY121768 molecule not only confers high selectivity for type A MAO but offers a site for radionuclide introduction that might be a useful means of labeling type A MAO in vitro or in vivo for various purposes.     

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.     

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

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.     

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.     

Genotoxicity of N-nitrosothiazolidine in microbial and hepatocellular test systems

The genotoxicity of N-nitrosothiazolidine was studied in a microbial system and in a hepatocellular system. The compound showed positive responses in both tests, exhibiting weak mutagenicity at the lowest level tested (1 mg/plate) in the rec assay in Bacillus subtilis, and inducing statistically significant levels of DNA repair in primary hepatocyte cultures at concentrations ranging from 2 × 10^?4 to $\text{2 × 10}{}^{\mathrm{?3}}\text{m}$.     

Electrocortical changes induced by perfusion of catecholamines into the brainstem reticular formation

Various concentrations of noradrenaline and related catecolamines were perfused bilaterally into the pontine and mesencephalic reticular formation using push-pull cannulae. The initial effect of a 5-min perfusion of (-)-noradrenaline, (-)-adrenaline, (-)-phenylephrine or α-methyl noradrenaline, was to induce a two stage change in the electrocortical activity, that is, phasic followed by tonic desynchronisation. When high concentrations of these substances were used, a secondary sedative effect correlated with the appearance of slow wave (2–4 Hz) activity in the electrocorticogram was observed. In contrast, (-)-isoproterenol, even in high concentrations, did not produce secondary sedative effects.Using tritium labelled (±)-[7-³H]-noradrenaline in the 10^?6 to 10^?3m concentration range, it was established that the total amount of drug diffusing into the brain tissue was very low. The appearance of phasic electrocortical changes correlated with 0·4–25 ng noradrenaline (base) within the brain at the end of each cannula. Tonic electrocortical desynchronization which appeared when $\text{9 × 10}{}^{\mathrm{?5}}\text{m}\text{to}\text{7 × 10}{}^{\mathrm{?4}}\text{m}$ solutions were used, gave tissue levels of exogenous noradrenaline (base) of 6·5–103 ng. Secondary sedative effects were usually observed with tissue levels in excess of 98 ng at the end of each cannula.     

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.     

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.     

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].     

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 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.     

Inhibition of catecholamine uptake and retention in synaptosomal preparations by tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids

The effects of racemic mixtures of representative tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids on the mechanisms of catecholamine uptake and retention were studied in synaptosomal preparations from whole rat brain. The synaptosomes were incubated with $\text{(}{}^{14}\text{C}\text{)-d,l-}\text{norepinephrine}$ or (¹⁴C)-dopamine in the presence of various concentrations of salsolinol (SAL), tetrahydropapaveroline (THP), 2,3,10,11-tetrahydroxyberbine (THB), or 2,3,10,11-tetramethoxyberbine (TMB). Levels of radioactivity in synaptosomes preloaded with labeled norepinephrine were significantly diminished by the addition of 10^?4 M THP, THB or SAL to approximately 42.7 per cent (P < 0·001), 85.8 per cent (P < 0·02) and 85.9 per cent (P < 0·01), respectively, of control preparations. THP, 10^?5 M, also significantly decreased synaptosomal retention of the labeled neuroamine. (¹⁴C)-dopamine was used in an analysis of alkaloid effects on catecholamine uptake kinetics. K_i values obtained were: 0·7 × 10^?5 M (THP); 3·5 × 10^?5 M (THB); 1·25 × 10^?4 M (SAL); and 2·2 × 10^?4 M (TMB). These results have been interpreted to suggest that the affinity of these amine-derived alkaloids for the catecholaminergic uptake mechanisms, although not marked when compared to that of dopamine or norepinephrine, may be sufficient under conditions of highly localized formation and accumulation to have important physiological sequelae.