Evidence for saturation of catechol-0-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/1) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min^–1 and 0.186 min^–1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline.In isolated lungs perfused with ³H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l ³H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake₁ of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because V_{max uptake} V_{max COMT} for the metabolizing system consisting of non-neuronal uptake₁ + COMT in the lungs, as has been described previously for the system consisting of uptake₂ + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake₁, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake₁. In addition, another drug that is both a substrate of uptake₁ and a MAO inhibitor, debrisoquine, was investigated in the study.The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake₁ in rat lungs. After loading of isolated perfused lungs with ³H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of ³H-noradrenaline was measured for 30 min. When 1 mol/l amezinium or 15 mol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of ³H-noradrenaline, which was reduced by about 70% when 1 mol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l ³H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l ³H-noradrenaline under conditions where the amine was accumulated by uptake₂ (COMT, uptake₁ and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline.The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake₁. Amezinium is much less potent as a MAO inhibitor in cells with the uptake₂ transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.Abbreviations COMT catechol-O-methyltransferase - DOMA 3, 4-dihydroxy-mandelic acid - DOPEG 3, 4–'dihydroxyphenylglycol - ECS extracellular space - FRL fractional rate of loss - IC ₅₀ inhibitor concentration that causes 50% inhibition - K _{m uptake} Michaelis or half-saturation constant for uptake - k _{M AO} rate constant for deamination - k _{out NA} rate constant for efflux of noradrenaline - MAO monoamine oxidase - MAO-Aa type A monoamine oxidase - MAO-B type B monoamine oxidase - T/M _NA tissue to medium ratio of noradrenaline - U-0521 3, 4-dihydroxy-2-methylpropiophenone - V _max maximal rate - v _st–st steady-state rate of metabolite formation Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Bryan-Lluka 1993).     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A     

Different roles for type A and type B monoamine oxidase in regulating synaptic dopamine at D-1 and D-2 receptors associated with adenosine-3′,5′-cyclic monophosphate (cyclic AMP) formation

Summary The roles of multiple forms of monoamine oxidase (MAO) in regulating the synaptic concentration of dopamine, in the vicinity of dopamine receptors associated with cyclic AMP formation, was examined in striatal slices of the rat. d-Amphetamine (0.1 mol/l to 20 mol/l) caused a concentration-related increase in cyclic AMP formation, which correlated (in superfusion experiments) with the release of endogenously-formed dopamine. In the presence of (–)sulpiride (50 pmol/l), cyclic AMP formation was significantly increased at every concentration of d-amphetamine tested. At the same time, this concentration of (–)sulpiride had no effect on DA release. Inhibition of type A MAO with clorgyline (0.1 mol/l) significantly enhanced the increase in cyclic AMP formation seen after d-amphetamine. By contrast, inhibition of type B MAO with deprenyl (0.1 mol/l) was without effect on this action of d-amphetamine. At high concentrations of d-amphetamine (20 mol/l), however, deprenyl + clorgyline treatment enhanced cyclic AMP formation to a greater extent than with clorgyline alone. Similar results could be obtained at lower concentrations of d-amphetamine (5 mol/l), but only after inhibition of the dopamine neuronal reuptake system with nomifensine (30 gmol/1). Furthermore, in the presence of nomifensine, deprenyl alone was also able to significantly increase the cyclic AMP formation seen after d-amphetamine (5 gmol/l). In the presence of (–)sulpiride, relatively similar results were obtained following all MAO inhibitor treatments. These findings support the notion that type A MAO plays the primary role in regulating dopamine concentrations at D-1 and D-2 receptors within synapses of rat striatal tissue. However, a minor role for type B MAO can be demonstrated when high synaptic concentrations of dopamine are achieved or dopamine deamination is restricted to postsynaptic sites. Experiments with amezinium (10 mol/l) were conducted to further explore the importance of presynaptic and postsynaptic type A MAO in the regulation of synaptic concentrations of dopamine; total type A MAO inhibition was assessed with clorgyline (0.1 mol/l). caused a two-fold increase in the ability of d-amphetamine (5 mol/l) to increase cyclic AMP formation. This result represented 70% of the response obtained after total type A MAO inhibition with clorgyline. The values obtained with clorgyline, but not amezinium, were potentiated by inhibition of the dopamine neuronal reuptake system with nomifensine. These results support the idea that presynaptic type A MAO is highly instrumental in regulating synaptic dopamine concentrations; possibly by regulating the size of the pool of releasable dopamine. Postsynaptic type A MAO, however, can also play an effective role in the regulation of synaptic DA levels in instances where the dopamine neuronal reuptake system may become compromised.Abbreviations DA dopamine - CAMP cyclic AMP; adenosine-3,5-monophosphate - MAO monoamine oxidase Supported in part by the West Virginia University Medical Corporation, N. I. H. grant 5-T32-GM 07039, and a grant from the Fraternal Order of Eagles. Some of the findings were presented at the annual meeting of the Society for Neuroscience, Washington, DC (Azzaro and Liecione 1986) Send offprint requests to A. J. Azzaro     

Adenosine 3′,5′-monophosphate in cultured neuroblastoma cells: Effect of adenosine,phosphodiesterase inhibitors and benzazepines

Summary The effect of various neurohormones on intracellular levels of adenosine 3,5-monophosphate were evaluated in a neuroblastoma cell line both, in the presence and in the absence of the phosphodiesterase inhibitors isobutylmethylxanthine and papaverine. Without the phosphodiesterase inhibitors only prostaglandin E₁ increased intracellular adenosine 3,5-monophosphate levels. In the presence of isobutylmethylxanthine and/or papaverine, however, adenosine stimulated adenosine 3,5-monophosphate formation and the effect of prostaglandin E₁ was greatly potentiated. Treatment of the cells with dopamine, 5-hydroxytryptamine, noradrenaline, adrenaline, histamine and prostaglandin F₁ was without effect on adenosine 3,5-monophosphate levels either in the presence or absence of the phosphodiesterase inhibitors. The adenosine concentration for a half maximal effect was about 75 M. The effect of 0.1 mM adenosine was not antagonized by 1 mM theophylline. Several adenosine analogs were tested and found to have little or no effect on adenosine 3,5-monophosphate levels in neuroblastoma N4TG3. Diazepam and to a lesser extent chlordiazepoxide act like phosphodiesterase inhibitors when incubated together with prostaglandin E₁.Part of this work was done during a visit of the authors to NIH, U.S.A., J. S. being a fellow of the Deutsche Forschungsgemeinschaft and B. H. of the Max-Planck-Gesellschaft.     

Dopamine and adrenaline,but not isoprenaline,are substrates for uptake and metabolism in isolated perfused lungs of rats

Summary The uptake and subsequent metabolism by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) of dopamine, adrenaline, isoprenaline and noradrenaline in isolated perfused lungs of rats has been examined. In lung preparations in which COMT and MAO were inhibited, the uptake of ³H-labelled dopamine, (–)-adrenaline and (–)-noradrenaline, but not (±)-isoprenaline, was reduced by cocaine (10 or 100 mol/l) The rank order of the Km values of the amines that were substrates for uptake in the lungs were: dopamine (0.246 mol/l) < noradrenaline (0.967 mol/l) < adrenaline (3.32 mol/l). These results are consistent with transport of catecholamines in rat lungs by Uptake₁.In lung preparations with COMT and MAO intact, dopamine and noradrenaline were removed from the circulation (50% and 32%, respectively) and mainly metabolized. There was very little (3.0%) removal of isoprenaline by the lungs and adrenaline was not included in this part of the study. In lung preparations in which only MAO was inhibited, the rank order of COMT activity for O-methylation of the amines was dopamine noradrenaline adrenaline (k_COMT values: 4.98 min^–1, 0.357 min^–1, and 0.234 min^–1, respectively).If dopamine or adrenaline are perfused through the pulmonary circulation in isolated lungs of the rat, they are taken up and then metabolized by COMT and MAO, as also occurs for noradrenaline. Isoprenaline is not a substrate for uptake in the lungs. There was less uptake of adrenaline than noradrenaline, indicating that uptake and metabolism in the lungs may not be a significant removal process for adrenaline in the circulation of rats in vivo. The more marked uptake of dopamine (than of noradrenaline) indicates that uptake and metabolism by the lungs, at least in the rat, may play an important role in the removal of dopamine from the circulation in vivo.Abbreviations COMT catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPEG 3,4-dihydroxyphenylglycol - DOPET 3,4-dihydroxyphenyl ethanol - MAO monoamine oxidase - MN metanephrine - MTA 3-methoxytyramine - NMN normetanephrine - OMDA O-methylated deaminated metabolites - OMI 3-O-methylisoprenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone Some of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists (Bryan and O'Donnell 1987, 1988; Bryan et al. 1989; Bryan-Lluka 1990) Send offprint requests to L.J. Bryan-Lluka at the above address     

Hemmung von Adenyl-Cyclase-Präparationen aus der Rattenniere durch Calciumionen und verschiedene Diuretica

Summary Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 35-monophosphate (Ado-35-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-35-P formed from 1 mM ¹⁴C-ATP in the presence of 10 mM Mg⁺⁺, an ATP regenerating system, and 5 mM unlabeled Ado-35-P to reduce the enzymatic degradation of the labeled Ado-35-P.Calcium ions reduced the rate of Ado-35-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg⁺⁺ or Mn⁺⁺. With 10 mM Mg⁺⁺, 1 mM Ca⁺⁺ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca⁺⁺. The inhibition was independent of the ATP concentration, but was influenced by the Mg⁺⁺ content of the incubation medium.Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-35-P in the regulation of sodium transport is as yet unclear.Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration.The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 35-monophosphate phosphodiesterase, reduced the rate of Ado-35-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues.Instead of phosphodiesterase inhibitors, unlabeled Ado-35-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-35-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-35-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-35-P by 35%.

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Abkürzungen Ado-35-P Adenosin-35-monophosphat - Guo-35-P Guanosin-35-monophosphat - ADH antidiuretisches Hormon, Vasopressin - PTH Parathormon - TCT Thyreocalcitonin - DTE Dithioerythrit - EDTA Äthylendiamintetraessigsäure Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Über einen Teil der Ergebnisse wurde auf der 11. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft berichtet (Jakobs et al., 1970). Einige der vorliegenden Ergebnisse sind der Inauguraldissertation von K. H. J. (Medizinische Fakultät der Universität Heidelberg, 1971) entnommen.     

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain

Binding characteristics of a novel radioiodinated ligand, [¹²⁵I]R(+)trans-7-hydroxy-2-(N-n-propylN-3-iodo-2-propenyl) aminotetralin (¹²⁵I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [¹²⁵I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (K_d = 0.42 nM). A majority (70%) of the sites labeled by [¹²⁵I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to 5-HT_1A receptors, with a K_d value of 1.4 nM and a B_max value of 210 fmol/mg protein, was observed. Binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D 2 and 5-HT_1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [¹²⁵I]R(+)trans-7-OH-PIPAT. [¹²⁵I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D 3 receptors.Abbreviations 7-OH-DPAT 7-hydroxy-2-N,N-(di-n-propyl)aminotetralin - trans-7-OH-PIPAT traps-7-hydroxy-2-(N-n-propyl-N-3-iodo-2propenyl)aminotetralin - 8-OH-DPAT 8-hydroxy-2-N,N-(di-n-propyl)aminotetralin - 8-OH-PIPAT trans-8-hydroxy-2-(N-n-propyl-N-3-iodo-2-propenyl)aminotetralin - NCQ298 S(–)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy-5,6-dimethoxybenzamide - DTG 1,3-diortho-tolyl-guanidine - Sf9 cells Spodoptera frugiperda insect cells - CHO cells Chinese hamster ovary cells - GTP guanosine 5-triphosphate - WB4101 2-(2,6-dimeethoxyphenoxyethyl)aminomethyl-1,4benzodioxane - R(+)3PPP R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine - (+)MK-801 (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine     

Inhibition of monoamine oxidase and semicarbazide-sensitive amine oxidase by mexiletine and related compounds

Summary The in vitro inhibition by mexiletine and related compounds of the activity of rat brain, heart and lung mono-amine oxidase-A (MAO-A), rat brain MAO-B, human platelet-poor plasma benzylamine oxidase and a clorgyline-resistant, semicarbazide-sensitive amine oxidase (SSAO) distinct from both MAO and benzylamine oxidase has been studied. The compounds were most active towards MAO-A and SSAO. IC₅₀ values for mexiletine towards rat heart MAO-A and SSAO were 10 mol/l and 320 mol/l, respectively. Replacement of the para-hydrogen atom in the mexiletine aromatic ring by bromine increased potency towards both MAO-A and SSAO. Replacement of the ortho-methyl group in the mexiletine aromatic ring by hydrogen increased the potency towards SSAO alone. FLA 1042, with both these substitutions, was found to be a reversible mixed-type inhibitor of both MAO-A (K _i ^slope 1.4 mol/l, K _i ^int 24 mol/l) and of SSAO (K _i ^slope 12 mol/l, K _i ^int 6 mol/l).     

The Solution Conformations of Lidocaine Analogues

IR and ¹H NMR studies in CDCl₃ and CCl₄ of a series of tertiary aminoxylidides with the amino group in the 2 to 6 position of the acyl chain are described. Lidocaine, diethylaminoaceto-2,6-xylidide, forms an intramolecular five-membered ring hydrogen-bonded monomer at all concentrations in both solvents. -Diethyl-amino-propiono-2,6-xylidide forms an intramolecular six-membered ring hydrogen-bonded monomer in CDCl₃ and CCl₄ but a trans intermolecularly associated species is the major form present at high concentrations in CCl₄. The longer-chain homologues are mixtures of nonassociated trans and cis monomers at low concentrations but associated trans forms predominate at high concentrations. Evidence for the presence of a hydrogen-bonded seven-membered ring intramolecular monomer in CDCl₃ for -diethylaminobutyro-2,6-xylidide is presented. The relationship between the molecular conformation and the partition coefficient is discussed.     

Identification of a novel high affinity adenosine binding protein from bovine striatum

Summary In solubilized extracts from bovine striatal membranes three different binding sites for 5-N-ethylcarboxamidoadenosine ([³H]NECA) were observed after separation of the extract by gel filtration on Sepharose CL-6B. The first peak was eluted in the void volume and contained the AZ adenosine receptor. In the second peak, [³H]NECA binding sites were eluted with a pharmacological profile characteristic of adenotin, a low affinity non-receptor adenosine binding protein. The third peak represented approximately 50% of the [³H]NECA binding activity. This site bound [³H]NECA in a reversible and saturable manner with K _D of 17 nmol/l and a binding capacity of 11.3 pmol/mg protein. In competition experiments, adenosine, NECA, NAD, nnosine, 5-AMP and S-adenosyl-L-homocysteine were the most potent ligands. In contrast to adenosine receptors, this site did neither bind adenosine receptor antagonists nor the A₂ selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)phenethylamino]5-N-ethylcarboxamidoadenosine). These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine striatum.Abbreviations AMPPCP ,-methyleneadenosine-5-triphosphonate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - CGS 21,680 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarbox-amidoadenosine - CPA N⁶-cyclopentyladenosine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - GppNHp guanosine-5-[,-imido]triphosphate - GTP[S] guanosine-5-O-(3-thiotriphosphate) - NBTI S-4-nitrobenzyl-6-thioinosine - NECA 5-N-ethylcarbox-amidoadenosine - NECG 5-N-ethylcarboxamidoguanosine - PIA N⁶-phenylisopropyladenosine - SAH S-adenosyl-L-homocysteine - XAC 8-{4-[([{(2-aminoethyl)-amino}carbonyl]-methyl)oxy]-phenyl}-1,3-dipropylxanthine Send offprint requests to A. Lorenzen at the above address     

Trennung und Bestimmung der Nucleotide des Gehirns

Ohne ZusammenfassungFolgende Abkürzungen werden in der Arbeit verwendet AMP Adenosin-5-monophosphat - ADP Adenosin-5-diphosphat - ATP Adenosin-5-triphosphat - GMP Guanosin-5-monophosphat - GDP Guanosin-5-diphosphat - GTP Guanosin-5-triphosphat - IMP Inosin-5-monophosphat - UMP Uridin-5-monophosphat - UDP Uridin-5-diphosphat - UTP Uridin-5-triphosphat - UDPAG Uridin-5-diphosphat-N-acetylglucosamin - UDPG Uridin-5-diphosphat-glucose - DPN Diphosphopyridinnucleotid - TPN Triphosphopyridinnucleotid Mit 10 TextabbildungenMit Unterstütznng der Deutschen Forschungsgemeinschaft.     

Pharmacokinetics of sulfametopyrazine (Kelfizine W) effects of a single oral dose

Summary The pharmacokinetics of sulfametopyrazine were studied for seven days after a single oral dose of 2 g. in healthy volunteers in order to establish its chemotherapeutic value. — The appearance and disappearance of the drug in the plasma were evaluated both for compounds with a free amino group and for total sulphonamides. The half-life and absorption, distribution, elimination and excretion coefficients were calculated, as well as the concentrations in plasma water and interstitial fluid. The estimated drug concentrations in the urine agreed with those calculated from the excretion coefficients. — In all subjects at the end of the seventh day the concentrations in all body compartments of active compounds exceeded the minimum required for a therapeutic effect. The highest concentrations found in the urine were always significantly lower than the drug's basal solubility at pH 5, thus excluding any risk of crystalluria.Glossary of symbols total binding capacity of plasma proteins for SMP - Specific gravity of blood ( Bl), and interstitial fluid (IF) - minimum inhibitory concentration for bacterial growth. Evaluation of against E. coli or other pathogenic bacteria in a medium free of antagonists [29] - ratio of dose interval to half-life - dose interval - safety factor. Proportionality constant between andc _min for a therapeutic efficacy of 95 per cent - fraction of the administered drug absorbed from the depot (gastrointestinal tract etc.) - distribution coefficient with respect to the drug concentration in blood plasma (ml/g) - D* initial dose of the drug - D maintenance dose - M molecular weight of the drug (280) - G weight of subject (kg) - F area between time axis and concentration curve (plotted c' values) - t _50% apparent biological half-life - w ¹ water content of plasma (ml/ml) - p protein concentration of plasma (pBl), or interstitial fluid (pIF) (g/l) - c _IF concentration in the interstitial fluid - C ₀ concentration in plasma at zero time after i.v. administration - c ₁ ⁰ concentration in plasma after oral absorption extrapolated to zero time - c ₁ concentration in plasma water of the drug with free amino function - c _min minimum inhibitory concentration needed in plasma water (minimum therapeutic concentration) - k ₀₁ rate constant for absorption - k ₁ rate constant for absorption determined at timet; (similarlyk₂) - K total elimination coefficient - k _el rate constant for elimination - k _F rate constant for formation of metabolites - k _D excretion coefficient of SMP with free amino function - k _U coefficient of metabolite excretion - D ₀ quantity of SMP in the body at time zero - D _B quantity of SMP in the body at timet - D _U quantity of SMP excreted in the urine at timet - M _F quantity of metabolites formed at timet - M _B quantity of metabolites present in the body at timet - M _U quantity of metabolites excreted in the urine at timet - K dissociation constant for the sulphonamide-protein complex - notation for quantities related to drug concentrations in plasma, e.g. c (corresponding term without refer to plasma water)     

Lysergic acid diethylamide antagonizes shaking induced in rats by five chemically different compounds

Thyrotropin-releasing hormone (TRH), sodium valproate, AG-3-5 (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), RX336-M (7,8-dihydro-5, 6-dimethylcyclohex-5-eno-1,2,8,14 codeinone), and Sgd 8473 (-[(4-chlorobenzylideneamino)-oxy]-isobutyric acid) each induced repetitive shaking of the body of rats after intraperitoneal injection. This action of the five diverse chemicals appears to be subserved by a common pharmacological component, because pretreatment with d-lysergic acid diethylamide (0.03–1.0 mg kg^-1, s.c.) attenuated the shaking behavior in a dose-related manner, and cross tolerance was found between RX 336-M and TRH, sodium valproate, and AG-3-5.     

Comparative studies on distribution and covalent tissue binding of 2,4,2′,4′- and 3,4,3′,4′-tetrachlorobiphenyl isomers in the rat

The ¹⁴C-labeled tetrachlorobiphenyl (TCB) isomers 2,4,2,4-tetrachlorobiphenyl (2,4,2,4-TCB) and 3,4,3,4-tetrachlorobiphenyl (3,4,34-TCB) were administered orally to rats, and distribution and covalent binding were measured in several organs. Marked differences in distribution and covalent binding of the two TCBs were observed. The accumulation and retention of 2,4,2,4-TCB in adipose tissue were much higher than those of 3,4,3,4-TCB, although the level of radioactivity in the blood was consistently higher in 3,4,3,4-TCB treated rats. The radioactivity bound in covalent linkages with cellular macromolecules in several tissues was also measured. The data obtained indicated that covalent binding was higher in 3,4,3,4-TCB treated rats than in those treated with 2,4,2,4-TCB, particularly in liver and blood components. These results suggest that the two TCB isomers have different pharmacokinetic properties in rats, and the association of covalent binding with 3,4,3,4-TCB-induced toxicities might be important. In addition, we found that repeated oral dosing with the two TCB isomers caused an increase in in vitro liver microsomal generation of reactive metabolites of TCBs, indicating that the microsomal enzyme system is likely to play an important role in the in vivo covalent binding of TCB.     

Synthesis and transformations of furan derivatives

By the reactions of 2-phenyl-4-(2-furfuryliden)-, 2-phenyl-4-(5-nitro-2-furfuryliden)-, and 2-methyl-4-(5-nitro-2-furfuryliden)-5-oxazolones with primary and secondary amines, a series of N-mono- and N,N-disubstituted amides of the corresponding-benzamido--(2-furyl)-acrylic and-benzamido- and-acetamido--(5-nitro-2-furyl)acrylic acids was synthesized. 1-Alkyl(aryl) substituted 2-phenyl-4-(5-nitro-2-furfuryliden)-5-imidazolones were synthesized from the reaction of phosphorus oxychloride and the monosubstituted amides of-benzamido--(5-nitro-2-furyl)acrylic acid.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 2, pp. 21–27, February, 1967.     

Adrenocorticolytic DDT-metabolites: studies in mink,Mustela vison and otter,Lutra lutra

The irreversible binding and toxicity of the DDT metabolites p,p-DDD and o,p-DDD in the adrenal cortex of female mink Mustela vison were studied. Histological examination of adrenals from mink given a single i.p. injection of p,p-DDD or o,p-DDD (125 mg per kg body weight) showed vacuolation, necrosis and focal bleedings in the zonae fasciculata and reticularis. Autoradiograms of solvent-extracted tissue sections of minks given a single i.v. injection of p,p-[¹⁴C]DDD (0.7 mg per kg body weight) revealed a high level of irreversibly bound radio-activity in the adrenal cortex. Microautoradiography showed that the irreversibly bound radio-activity was confined to the zonae fasciculata and reticularis. Incubation of p,p-[¹⁴C]DDD and o,p-[¹⁴C]DDD with mink or otter, Lutra lutra, adrenal homogenate (300 x g supernatant) resulted in an irreversible binding of radioactivity to protein from both species. The irreversible protein binding of the DDD isomers in mink and otter was decreased by addition of the cytochrome P450 inhibitors metyrapone and carbon monoxide, indicating a cytochrome P450 dependent metabolic activation. In contrast, 3-methylsulfonyl-[¹⁴C]DDE, a potent adrenocortical toxicant in mice, does not appear to be metabolized to a reactive metabolite in the adrenal cortex of mink or otter. In conclusion, both p,p-DDD and o,p-DDD are toxic to the mink adrenal zona fasciculata and reticularis following activation in situ to reactive, tissue-binding metabolites. The results suggest that p,p-DDD and o,p-DDD are adrenocortical toxicants also in otter. The involvement of environmental pollutants in the generation of the adrenocortical hyperplasia observed among Baltic seals is discussed.Abbreviations p,p-DDD 2-(4-chlorophenyl)-2(4-chlorophenyl)-1,1-dichloroethane - o,p-DDD 2-(2-chlorophenyl)-2(4-chlorophenyl)-1,1-dichloroethane - MeSO₂-DDE 3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethane - DDT 2-(4-chlorophenyl)-2-(4-chlorophenyl)-1,1,1-trichloroethane - GSH reduced glutathione - PCB polychlorinated biphenyls