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%.

---

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.     

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     

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).     

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     

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3-dimethylbenzidine, and 3,3-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3-dimethoxybenzidine-based dye), Direct Black 38 (a benzidinebased dye), and Evans Blue (a 3,3-dimethylbenzidine-based dye) all bound to albumin, ₁-lipoprotein, -lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and ₁-antichymotrypsin, and degraded C₃ globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of ₁-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3-dimethylbenzidine to both ₁- and -lipoproteins.Supported by the Bal F. Swan Foundation, Denver, CO     

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.     

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.     

Toxicity and 7-ethoxyresorufin O-deethylase-inducing potency of coplanar polychlorinated biphenyls (PCBs) in chick embryos

The toxicities of the coplanar polychlorinated biphenyls 3,3,4,4-tetrachlorobiphenyl (TCB), 3,3,4,4,5-pentachlorobiphenyl (PeCB) and 3,3,4,4,5,5hexachlorobiphenyl (HCB) were compared in a 72-h study on chick embryos. The substances were injected into the air sacs of hens' eggs preincubated for 7 days. Mortality was measured 72 h later and corresponding LD₅₀ values were calculated. The rank order of toxicity was PeCB> TCB>HCB. Using the same injection procedure, the potencies of these chlorobiphenyls with regard to their induction of hepatic 7-ethoxyresorufin O-deethylase activity were compared. The ranking order of the substances as inducers was the same as their order when ranked according to toxicity. The three coplanar chlorobiphenyls were considerably more toxic and potent as inducers than the nonplanar 2,2,4,4,5,5-hexachlorobiphenyl. In a 2-week toxicity study, PeCB and HCB were injected into the yolks of hens' eggs preincubated for 4 days. PeCB was about 50-fold more potent than HCB in causing embryonic death. Both substances caused abnormalities, including edema, liver lesions, microphthalmia and beak deformities.     

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.     

Amine Prodrugs Which Utilize Hydroxy Amide Lactonization. I. A Potential Redox-Sensitive Amide Prodrug

Several amides of 3-(3,6-dioxo-2,4-dimethylcyclohexa-l,4-diene)-3,3-diniethylpropionic acid (2) have been synthesized and tested as model redox-sensitive pro-prodrugs of amines. The reduction of these model pro-prodrugs generated hydroxy amide intermediates 4a-4h, the lactonization of which resulted in amine release. The rates of lactonization of 4a-4h were investigated at pH 7.4 and 37°C. The half-lives for appearance of the product lactone la from these intermediates were found to range from 1.4 to 3.4 min. With such rapid lactonization rates, it is believed that reduction will be the rate-limiting step in the two-step conversion of the pro-prodrug to the amine.     

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.     

Evaluation of cardiotoxicity of a new anthracycline derivative: 4′ -deoxy-4′ -iodo-doxorubicin

Summary The present study in rats was performed to evaluate the cardiotoxic activity of 4 -deoxy-4 -iodo-doxorubicin (4-deoxy-4-I-DXR), a new anthracycline derivative with interesting antineoplastic properties and possible lower cardiotoxicity than doxorubicin (DXR). DXR produced ECG alterations consisting of a progressive and irreversible prolongation of SaT and QaT. In contrast, in 4-deoxy-4-I-DXR-treated rats the increase in SaT and QaT duration was significantly lower than that observed in DXR-treated rats and slightly increased over controls.DXR produced significant histologic changes in myocardium consisting of myocyte vacuolization and myofibrillar loss. No significant modifications were observed in mitochondria. In contrast, no significant cardiac lesions were observed in 4-deoxy-4-I-DXR-treated rats. These results suggest that this new anthracycline derivative has a significantly lower degree of cardiotoxic activity than DXR.     

Structure-activity relationship of covalently dimerized insulin derivatives: correlation of partial agonist efficacy with cross-linkage at lysine B29

The effects of 7 covalently dimerized insulin derivatives on glucose transport in differentiated 3T3-L1 cells were investigated. Symmetric cross-linkage at lysine B29 with a bridge of 2 (oxalyl), 8 (suberoyl) or 12 (dodecanedioyl) carbon atoms produced derivatives with essentially unaltered receptor binding affinity but largely reduced intrinsic activity. Regardless of the chain length, these derivatives inhibited the effect of submaximal insulin concentrations. Insulin derivatives cross-linked at phenylalanine 131 or asymmetrically at 131/1129 were full agonists of the insulin receptor. When lysine B29 was cross-linked with the inactive desoctapeptide(B23-B30)insulin at phenylalanine B1, the intrinsic activity of the resulting dimer was lower than that of insulin, but higher than that of the symmetric B29-dimers. It is concluded that linkage at the B29-lysines, and not at the B1-phenylalanine, leads to partial agonism of dimerized insulin derivatives, regardless of the length of the crosslinker.Abbreviations B29 ox-B29 dimer - B29,B29 oxalyl-(insulin)₂ - B29 sub-B29 dimer - B29,B29 suberoyl-(insulin)₂ - B29 dode-B29 dimer - B29,B29 dodecanedioyl-(insulin)₂ - B1-sub-B1 dimer, B1,B1 suberoyl-(insulin)₂ - B29-sub-B1 dimer, B29,B1 suberoyl-(insulin)₂ - B1(DOP) sub-B1dimer, 131(B23-B30-desoctapeptide)insulin-B1-suberoyl-insulin - B1(DOP) sub-B29 dimer, B1(B23–B30-desoctapeptide)insulin-B29-suberoyl-insulin     

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.     

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.     

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.     

Rat adrenal cyclic nucleotide-phosphodiesterase; Inhibition by drugs known to affect steroidogenesis

Summary A cyclic 3,5-nucleotide phosphodiesterase from rat adrenals was partially purified. The enzyme preparation had a pH optimum at 7.5, the activity being dependent on Mg²⁺, similar to the enzyme in other organs. Adenosine 3,5-monophosphate, guanosine 3,5-monophosphate and inosine 3,5-monophosphate were about equally well degradated (K _m 0.1 mM), while 2-O-deoxy-adenosine 3,5-monophosphate and tubercidin 3,5-monophosphate had a considerably higher K _m. In contrast to rat adipose tissue, rat adrenal phosphodiesterase did not hydrolyse uridine 3,5-monophosphate. Adrenal phosphodiesterase was inhibited competitively by methylxanthines, papaverine and eupaverin, eupaverin being the most potent inhibitor. Adenosine, its phenylisopropyl-analogue and metabolic products of adenosine inhibited adrenal phosphodiesterase, but were considerably less potent than methylxanthines or papaverine. All inhibitors tested are able to affect either spontaneous and/or stimulated synthesis of corticosteroids in rat adrenals as shown elsewhere. The data obtained with adrenal phosphodiesterase do not allow the conclusion that inhibition of this enzyme can be correlated with effects on steroidogenesis.Part of this work has been presented at the 12. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (Klotz et al., 1971).     

Der Einfluß von Hydrochlorothiazid und anderen sulfonamidierten Diuretica auf die 3′,5′-AMP-Phosphodiesterase-Aktivität in der Rattenniere

Summary Vasopressin has been reported to accelerate the conversion of adenosine triphosphate to cyclic 3,5-AMP by stimulating the activity of the adenyl cyclase. According to the view of Orloff and Handler cyclic 3,5-AMP is responsible for the augmentation of osmotic water flow. The cyclic nucleotide ist degraded by the enzyme 3,5-AMP phosphodiesterase (PDE) to 5-AMP. Inhibition of this enzyme by theophylline results in an increase in the concentration of 3,5-AMP and a concomittant increase in osmotic water flow, as shown in the urinary bladder of the toad (Bufo marinus).The experiments presented in this paper derived from a previous observation that furosemide and hydrochlorothiazide inhibit PDE. Both diuretics have been shown to reduce renal PDE activity when injected i.v. to rats in a dose of 25 mg/kg. Following injection of furosemide PDE activity has been found reduced only in the cortex, the effect of hydrochlorothiazide has been shown to be restricted to the inner medulla.Studies on the subcellular distribution of renal PDE revealed two fractions, one third of total activity bound to large particles, probably cell membranes, two third soluble in the hyaloplasm. The two fractions of the enzyme differ in their k _m-value for 3,5-AMP. Subcellular distribution and k _m-values of PDE in the liver have been found to be identical with those in the kidney.Hydrochlorothiazide has been shown to affect both fractions of renal PDE. Because of the restriction of the action of furosemide to the renal cortex no attempt was made to differentiate the effect of the compound with respect to its subcellular localization. Accumulation of 3,5-AMP caused by an impaired degradation of the nucleotide in this region could lead to an increase in the permeability of the distal convoluted tubules to water. As the difference in the osmotic pressure between distal tubular fluid and the surrounding interstitial fluid is relatively small, the increase in water permeability can only result in a small increase in tubular water reabsorption. In view of hydrochlorothiazide reducing PDE activity in the inner medulla and the high difference in the osmotic pressure between the fluid in the collecting tubules and the interstitial fluid it is suggested that a hydrochlorothiazide induced increase in water permeability results in a high increase in water rea-absorption, especially in diabetes insipidus where there is a low osmolarity of the tubular fluid in the collecting duct with an unimpaired cortico-papillary osmotic gradient. This corresponds to the paradoxical antidiuretic effect of diuretics in the treatment of diabetes insipidus centralis and renalis, especially after diuretic induced sodium depletion and reduction of the osmolarity of tubular fluid resulting in an increased osmotic difference between fluids within collecting ducts and interstitium.

Am 31. Oktober 1967 verstorben.     

Pharmacological actions of the main metabolites of dihydroergotamine

Summary Dihydroergotamine (DHE) and 5 of its main metabolites, namely 8-hydroxy-dihydroergotamine (8-OH-DHE), 8,10-dihydroxy-dihydroergotamine (8,10-OH-DHE), 2,3seco,N(1)formyl,3-keto,8-hydroxy-dihydroergotamine (8-OH,N(1)formyl-DHE), dihydrolysergic acid amide (DH-LSA) and dihydrolysergic acid (DH-LS) were investigated on human and canine veins in vitro, on canine veins in situ, and in the ganglion-blocked rat in vivo. Like DHE, the metabolites 8-OH-DHE, 8,10-OH-DHE and DH-LSA caused contriction of human varicose veins and only weak -adrenoceptor blockade. On canine femoral vein strips the same compounds produced predominantly -adrenoceptor blockade and only negligible stimulation. 8-OH,N(1)formyl-DHE and DH-LS were largely inactive. The same compounds, which were agonists on human vein strips in vitro, induced dose-dependent reduction of venous compliance when infused locally into the dog saphenous vein in situ. In the ganglion-blocked rat, only 8-OH-DHE and 8,10-OH-DHE besides the parent drug produced an increase in diastolic blood pressure when injected intravenously. It is concluded that DHE metabolites with considerable venoconstrictor activity may contribute to the selective therapeutic action of DHE.     

The influence of 8-Br 3′,5′-cyclic nucleotide analogs and of inhibitors of 3′,5′-cyclic nucleotide phosphodiesterase,on noradrenaline secretion and neuromuscular transmission in guinea-pig vas deferens

Summary The effects of 3,5-cyclic nucleotide phosphodiesterase (PDE) inhibitors and of 8-Br 3,5-cyclic nucleotide analogs on nerve-muscle transmission were studied in the guinea-pig vas deferens preincubated with ³H-noradrenaline.8-Br cyclic AMP and the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and 3-propionyl-4-hydrazinopyrazolopyridine (SQ 20006) enhanced the secretion of ³H-NA evoked by transmural nerve stimulation. 8-Br cyclic GMP was without effect in this respect.The muscle contraction evoked by transmural nerve stimulation, high potassium or by application of exogenous noradrenaline was depressed by IBMX and SQ 20006. The contraction evoked by transmural nerve stimulation was enhanced by 8-Br cyclic AMP and depressed by 8-Br cyclic GMP.These findings suggest differential involvement of 3,5-adenosine- and guanosine-cyclic nucleotides in excitation-secretion-coupling in the noradrenergic sympathetic nerves, and in excitation-contraction-coupling in the smooth muscle, of guinea-pig vas deferens.