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.     

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     

Wirkung von cyclischem Adenosin-3′,5′-Monophosphat (3′,5′-AMP) und seinem Dibutyrylderivat (DBA) auf Lipolyse,Glykogenolyse und Corticosteronsynthese

Zusammenfassung 1. Am isolierten Fettgewebe von Ratten hatte das Dibutyrylderivat des cyclischen Adenosin-3,5-Monophosphat (DBA) eine etwa 100 mal stärkere lipolytische Wirkung als das nicht substituierte cyclische Adenosin-3,5-Monophosphat (3,5-AMP). Hormone (ACTH, Noradrenalin) waren an diesem Testobjekt 10000 mal wirksamer als DBA. Durch Hemmung der Phosphodiesterase mit Theophyllin ließ sich auch die Wirkung des DBA verstärken.2. An isolierten Nebennieren von Ratten stimulierte DBA die Corticosteronsynthese etwa 100 mal stärker als 3,5-AMP; ACTH war aber 500 mal wirksamer als DBA. Durch Theophyllin ließ sich die Wirkung von ACTH, DBA bzw. 3,5-AMP nicht verstärken. Hohe Konzentrationen des Xanthinderivates hemmten die Corticosteronsynthese.3. An Ratten war die hyperglykämische Wirkung des DBA wesentlich stärker als diejenige des 3,5-AMP: Für eine Erhöhung des Blutzuckerspiegels um 40 mg/100 ml benötigten wir von DBA weniger als 1 mol/kg, von 3,5-AMP aber 30 mol/kg. Diese Wirkung der Nucleotide ließ sich durch Theophyllin nicht verstärken. Der Fettsäuren- und Glyceringehalt des Plasmas wurde durch Injektion von DBA bzw. 3,5-AMP nicht erhöht, sondern erniedrigt. — Die Ergebnisse wurden im Zusammenhang mit dem Second Messenger Concept von Sutherland u. Mitarb. diskutiert.Über einen Teil der Ergebnisse wurde auf der 8. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (Stock u. Westermann, 1967; Bieck u. Westermann, 1967) sowie in einer kurzen Mitteilung (Bleck et al., 1968) berichtet.     

Comparative effects of adenosine and adenine and guanine nucleotides on drug biotransformation in rats

Summary The effect of various nucleotides and adenosine on the hepatic biotransformation of hexobarbital sodium (HB) and p-chloro-N-methylaniline (PCMA) was determined in male rats. Intraperitoneal administration of dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP), alone and in combination with theophylline, and the cyclic adenosine 3,5-monophosphate (cAMP)-theophylline combination prolonged HB sleeping time by more than 70%. cAMP or dibutyryl cyclic guanosine 3,5-monophosphate (DBcGMP), alone or in combination with theophylline, failed to significantly alter the duration of HB-induced hypnosis. Plasma levels of HB upon awakening suggested that the increase in sleeping time was apparently not due to an altered sensitivity of the brain to the barbiturate. The effect appears to be related to an impairment of HB metabolism since only those compounds that inhibited HB oxidation when added to liver slices prolonged hypnosis. In addition, 5-AMP, adenosine, and cyclic guanosine 3,5-monophosphate (cGMP) failed to alter HB biotransformation by liver slices. A similar pattern of impairment of metabolism by liver slices was observed when PCMA was used as the substrate. The inhibitory effect of DBcAMP was not altered by concurrent addition of either cGMP or DBcGMP. No impairment in the rate of microsomal PCMA biotransformation resulted from addition of adenosine or any of the nucleotides used in this study.     

Localization of Purine Metabolizing Enzymes in Bovine Brain Microvessel Endothelial Cells: An Enzymatic Blood-Brain Barrier for Dideoxynucleosides?

Purpose. The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (K_m and V_max) of purified calf spleen PNP for inosine and 2,3-dideoxyinosine (ddl) and of purified calf intestinal ADA for 2,3-dideoxyadenosine (ddA), 6-chloro-2,3-dideoxypurine (6-Cl-ddP), and 2--fluoro-2,3-dideoxyadenosine (F-ddA) have been explored. Methods. BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, -glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest. Results. The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex. Conclusions. ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddl, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.     

Cardiostimulatory effects of cyclic 3′,5′-adenosine monophosphate and its acylated derivatives

Summary The effects of cyclic 3,5-AMP and of two acylated derivatives, dibutyryl (DBA) and dihexanoyl-3,5-AMP (DHA) were investigated in isolated perfused hearts of guinea pigs, rats and rabbits.In guinea pig hearts, DBA (Ca- and Na-salt) and DHA-Na in high doses (10 moles) produced strong and long lasting increases in the rate and amplitude of contractions, coronary flow, and moderate increases in phosphorylase activity in the majority of experiments. The positive ino- and chronotropic effects occured 3–5 min after injection of the drug, mostly in a fluctuating manner with several maxima. Theophylline augmented the effects of DBA-Na and revealed positive inotropic actions of non substituted 3,5-AMP.In rat hearts, similar, but more pronounced and dose-dependent effects were observed after 1, 5 and 10 moles DBA-Na. Propranolol (50 g) did not block the action of 10 moles DBA-Na. Non substituted 3,5-AMP, 5-AMP and ATP in doses of 10 moles had no significant positive inotropic effects.In rabbit hearts, DBA-Na (50 moles) produced moderate, non fluctuating rises in the amplitude of contraction.The results provide evidence that under certain conditions cyclic 3, 5-AMP itself, like its acylated derivatives DBA and DHA, may produce strong and direct positive inotropic and chronotropic effects in the heart. These findings support the view that cyclic 3,5-AMP is the cellular mediator of the cardiostimulant actions of substances that increase its rate of production in the myocardial cell.The excellent technical help of Mrs. Vera Bauer is gratefully acknowledged by the authors.     

Rat diaphragm cyclic nucleotide-phosphodiesterase: Influence of drugs affecting skeletal muscle contractility

Summary In the present study a phosphodiesterase was partly purified from rat diaphragm and its properties as well as the effects of some drugs known to affect neuromuscular transmission were examined.The enzyme preparation had a pH optimum of 7.0–8.0 As for phosphodiesterase of other organs, the activity was dependent on Mg²⁺ and mainly located in the 100 000×g supernatant. It showed two apparent K _m values (6.4 and 390 M) for the cyclic adenosine-3,5-monophosphate hydrolysis. Various drugs inhibited diaphragm phosphodiesterase non-competitively in the following order of potency: eupaverine papaverine > 1-hexyl-3,7-dimethylxanthine > Ro 7-2956 > theophylline > d-tubocurarine > hydrochlorothiazide. Succinylcholine was ineffective.Of the cyclic nucleotides tested here only cyclic guanosine-3,5-monophosphate elicited an inhibiton at low concentrations (K _i=7M), while cyclic inosine-3,5-monophosphate and cyclic N₆-2-O-dibutyryl-adenosine-3,5-monophosphate inhibited only in high concentrations. Cyclic uridine-3,5-monophosphate did not inhibit phosphodiesterase. The type of inhibition was apparently competitive for cyclic N₆-2-O-dibutyryl-adenosine-3,5-monophosphate and cyclic guanosine-3,5-monophosphate, and non-competitive for cyclic inosine-3,5-monophosphate.The present findings on phosphodiesterase inhibitors agree well with our earlier results on the ability of these drugs (except d-tubocurarine) to increase muscular contractility. It is suggested that their mode of action might be facilitation of the release of acetylcholine from motor nerve endings via the accumulation of cyclic adenosine-3,5-monophosphate.     

Studies on the constituents of Scutellaria species (XXI): constituents of the leaves of Scutellaria strigillosa Hemsley

From the leaves of Scutellaria strigillosa, 14 compounds, chrysin, apigenin, 5,7,2-trihydroxyflavone, norwogonin, ursolic acid, 6-hydroxy-4-stigmasten-3-one, 6-hydroxy-4,22-stigmastadien-3-one, 2 R,4 R,8 R--tocopherol, (S)-5,5 -bi--tocopheryl, (R)-5,5 -bi--tocopheryl, solanachromene, tocopherylquinone, jodrellin T, and 14,15-dihydrojodrellin T were isolated. The structures were determined on the basis of chemical and spectral data.     

Antitumor Agents. 125. New 4β-Benzoylamino Derivatives of 4′-O-Demethyl-4-desoxypodophyllotoxin and 4β-Benzoyl Derivatives of 4′-O-Demethylpodophyllotoxin as Potent Inhibitors of Human DNA Topoisomerase II

A series of 4-benzoylamino (5-17) derivatives of 4-O-demethyl-4-desoxypodophyllotoxin and 4-benzoyl (18-20) derivatives of 4-O-demethyl podophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 5-13 and 15-17 are more potent than etoposide in causing DNA breakage, while compounds 9, 10, 13, 14, 16, and 20 are more active than etoposide in their inhibition of the human DNA topoisomerase II. The order for the enzyme inhibitory activity of the derivatives of 4-O-demethyl-4-desoxypodophyllotoxin is 4-arylamino > 4-benzylamino > 4-benzoylamino.     

Relaxation of hormonally stimulated smooth muscular tissues by the 8-bromo derivative of cyclic GMP

Summary Substances that cause contraction or relaxation of smooth muscle have been shown to increase intracellular levels of cyclic GMP. Because of the unclear role of cyclic GMP in the control of smooth muscle tone, cyclic GMP derivatives were exogenously applied to various smooth muscle preparations and their effects on tissue tone were studied.Whereas the basal tone of the rat ductus deferens was not affected by exogenous cyclic GMP or its dibutyryl or 8-bromo derivatives, the contractile responses of this tissue to noradrenaline and acetylcholine were depressed by preincubation with 10 M 8-bromo cyclic GMP (Br-cGMP). The 8-bromo derivatives of 2:3-cyclic GMP, 5-GMP and guanosine were without effects. Cyclic AMP levels were not changed by Br-cGMP. The frequency of oxytocin-stimulated rat uteri was also depressed by Br-cGMP (10 M). In helical strips of rat and rabbit aortae, Br-cGMP (1–100 M) caused a concentration-dependent, rapid decrease in noradrenaline-stimulated tissue tension. Br-2:3-cyclic GMP was ineffective. Noradrenaline-stimulated strips from hog spleen arteries were less sensitive to Br-cGMP than aortic tissue. In ductus deferentes and aortic strips stimulated by K⁺ at a depolarizing concentration, Br-cGMP caused less relaxation than under hormonal stimulation.These findings support the concept that cyclic GMP is involved in the control of smooth muscle tone and that hormone- and drug-induced elevations of the cyclic GMP level can reduce contractile responses to neurotransmitters and hormones.Abbreviations cGMP Guanosine 3:5-monophosphate, cyclic GMP - dibutyryl cGMP N², 2-O-dibutyryl guanosine 3:5-monophosphate - Br-cGMP 8-bromo guanosine 3:5-monophosphate - Br-2:3-cGMP 8-bromo guanosine 2:3-monophosphate - Br-GMP 8-bromo guanosine 5-monophosphate - Br-Guo 8-bromo guanosine, Br-guanosine - cAMP adenosine 3:5-monophosphate, cyclic AMP - dibutyryl cAMP N⁶, 2-O-dibutyryl adenosine 3:5-monophosphate - Br-cAMP 8-bromo adenosine 3:5-monophosphate This work was supported by grants from the Deutsche Forschungsgemeinschaft. Preliminary reports were presented (Schultz, 1977b; Schultz et al., 1978).     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria     

Dose-dependent excretion of DDE(1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in rats

Dose-dependent excretion of p,pDDE in rats was investigated. p,pDDE itself was the major excreta in rats. But some o,p'isomer of DDE was detected in feces by GC-MS analysis. The excretion of p,p'DDE after a single administration was modified by its dose level.The time pattern of p,pDDE excretion agrees well with the modified Hill equation. The value of the equilibrium constant (K) increases in proportion to time t after p,pDDE administration.Using the modified Hill equation and the linear K equation, the excretion rate of p,pDDE during the experimental time t can be estimated. The estimated p,pDDE excretion rate in feces agrees well with the measurements.     

Human urinary excretion of doxifluridine and metabolites during a 5-day chemotherapeutic schedule using fluorine-19 nuclear magnetic resonance spectrometry

The urinary excretion of doxifluridine (5 dFUrd) and its metabolites was determined during five days of chemotherapy using fluorine-19 nuclear magnetic resonance spectrometry. The daily urinary excretion of 5 dFUrd and its metabolites was high (-100% of the 5 dFUrd administered) and nearly constant through out the treatment. By far the major excreted compounds were unchanged 5 dFUrd and -fluoro--alanine which made up respectively -40% and -50% of the total. Neither accumulation of 5 dFUrd nor significant modifications in its metabolism were observed during the treatment.     

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     

Toxicokinetic interactions between chlorinated aromatic hydrocarbons in the liver of the C57BL/6J mouse: I. Polychlorinated biphenyls (PCBs)

2,2,4,4,5,5- (PCB 153), 2,3,3,4,4,5- (PCB 156) and 3,3,4,4,5,5-hexachlorobiphenyl (PCB 169) were administered orally to three groups of C57BL/6J mice using single doses of 1.5–109.1 mg/kg. Two other groups of mice received binary mixtures of PCB 153 and 156 or PCB 153 and 169. The hepatic deposition, elimination, CYP1a and CYP2b dependent enzyme activities were studied during a 77-day period. Some interactive effects on hepatic deposition and elimination were observed, resulting in increased deposition and faster elimination. These effects were most pronounced for the PCBs 156 and 169. A potentiating effect on hepatic CYP1a dependent 7-ethoxyresorufin-O-deethylation (EROD) activity was observed for the combination of PCB 156 and 153. Based on the results from the present study and earlier studies, it is suggested that the potentiating effect on EROD activity might be caused by a mechanism that is governed by at least two factors. The first is a toxicokinetic modulation of hepatic retention. The second factor is probably an elevation of hepatic Ah receptor levels by PCB 153.     

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.     

Phosphate and thiophosphate group donating adenine and guanine nucleotides inhibit glibenclamide binding to membranes from pancreatic islets

Summary In microsomes obtained from mouse pancreatic islets, the Mg complex of adenosine 5-triphosphate (MgATP) increased the dissociation constant (K _D) for binding of [³H]glibenclamide by sixfold. In the presence of Mg²⁺, not only ATP but also adenosine 5-0-(3-thiotriphosphate) (ATPS), adenosine 5-diphosphate (ADP), guanosine 5-triphosphate (GTP), guanosine 5-diphosphate (GDP), guanosine 5-0-(3-thiotriphosphate) (GTPTS) and guanosine 5-0-(2-thiodiphosphate) (GDP S) inhibited binding of [³H]glibenclamide. These effects were not observed in the absence of Mg²⁺. Half maximally effective concentrations of the Mg complexes of ATP, ADP, ATPS and GDP were 11.6, 19.0, 62.3 and 90.1 mol/l, respectively. The non-hydrolyzable analogues adenosine 5-(,-imidotriphosphate) (AMP-PNP) and guanosine 5-(,-imidotriphosphate) (GMP-PNP) did not alter [³H]glibenclamide binding in the presence of Mg²⁺. MgADP acted much more slowly than MgATP and both MgADP and MgADP did not inhibit [³H]glibenclamide binding when the concentrations of MgATP and MgATP were kept low by the hexokinase reaction. Development of MgATP-induced inhibition of [³H]glibenclamide binding and dissociation of [³H]-glibenclamide binding occurred at similar rates. However, the reversal of MgATP-induced inhibition of [³H]glibenclamide binding was slower than the association of [³H]glibenclamide with its binding site. Exogenous alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [³H]glibenclamide binding. MgATP enhanced displacement of [³H]glibenclamide binding by diazoxide. The data suggest that sulfonylureas and diazoxide exert their effects by interaction with the same binding site at the sulfonylurea receptor and that protein phosphorylation modulates the affinity of the receptor.Some of the results described here are part of the medical theses of S. Löser and I. Rietze Send offprint requests to M. Schwanstecher at the above address     

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.     

Identification of the Diastereomers of Pentobarbital N-Glucosides Excreted in Human Urine

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (lRS,5RS)-l--D-Glucopyranosyl) pentobarbital ((lRS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (lRS,5R)-PTBG and (lRS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/ MS). A HPLC method was developed for detecting and quantifying (lRS,5R)-PTBG, (lRS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6–6.2% of the pentobarbital dose was excreted as (lRS,5S)-PTBG over 60 hours. (lRS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (lR,5R)-PTBG and (lS,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.     

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.