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     

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.     

Cyanine-related compounds: a novel class of potent inhibitors of extraneuronal noradrenaline transport

Summary The neurotransmitter noradrenaline is removed from the extracellular space by neuronal and extraneuronal transport mechanisms. In the past, further functional and biochemical characterisation of the corticosterone-sensitive extraneuronal transporter was hampered by the lack of highly potent inhibitors. Here we describe a new class of selective and highly potent inhibitors of the extraneuronal noradrenaline transporter.Clonal Caki-1 cells possess the human type of extraneuronal noradrenaline carrier. The effect of various steroids and steroid-like compounds on initial rates of specific ³H-noradrenaline transport in Caki-1 cells was investigated. None of these steroids had an inhibitory potency higher than that of corticosterone which hitherto was generally accepted as the most potent inhibitor of the extraneuronal noradrenaline transport. On the other hand, a variety of quinoline and isoquinoline derivatives interacted with the extraneuronal noradrenaline transporter. Several cationic quinolines that belong to the chemical class of the cyanine dyes turned out to be very potent inhibitors of ³H-noradrenaline transport in Caki-1 cells. The isocyanines, 1,1-diisopropyl-2,4-cyanine (disprocynium24) and 1-methyl-1-isopropyl-2,4-cyanine as well as the pseudoisocyanines 1,1-diethyl-2,2-cyanine (decynium22) and 1-isopropyl-l-ethyl-2,2-cyanine (iprecynium22) were most potent with IC₅₀'s of 14, 62, 16, and 18 nmol/l, respectively. The inhibitory potency on extraneuronal noradrenaline transport of 1-methyl-l-isopropyl-2,4-cyanine was determined also in isolated organs, namely the isolated incubated rabbit aorta and the isolated perfused rat heart. The IC₅₀'s were 740 and 100 nmol/l, respectively. By contrast, the desipramine-sensitive neuronal type of noradrenaline transporter in PC 12 cells was hardly affected by the cyanine-related compounds. Decynium22 (3 mol/l) inhibited the neuronal noradrenaline transporter of clonal PC12 cells by 14% only.Cyanine-related compounds potently and selectively inhibit the extraneuronal transport mechanism for noradrenaline. They are expected to facilitate the functional and biochemical characterisation of the extraneuronal noradrenaline transporter.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) and the Universitätsbund Würzburg Correspondence to: H. Russ at the above address     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether ⁴⁵Ca²⁺ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC₅₀ = 45 M), ATPTS (EC₅₀ = 50 M) and 2-meSATP (EC₅₀ = 81 M) but not meATP (I mM) stimulated ⁴⁵Ca²⁺ influx 2–5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADPS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC₅₀ = 191 M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells.ATP-stimulated ⁴⁵Ca²⁺ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated ⁴⁵Ca²⁺ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC₅₀ = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.A number of P2 purinoceptor antagonists inhibited ATP-stimulated ⁴⁵Ca²⁺ influx. Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (3–300 M), pyridoxal 5phosphate (3–300 M) and d-tubocurarine (30–300 M) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC₅₀. Suramin (100–300 M) and cibacron blue (30–300 M) produced a surmountable antagonism while DIDS (4,4-diisothiocyana-tostilbene-2,2disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X₂ purinoceptors. These findings suggest that ATP-stimulated ⁴⁵Ca²⁺ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.     

Purification and characterization of an adenotin-like adenosine binding protein from human platelets

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.30% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL 6 B gel filtration, DEAE-Sepharose CL 6 B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5-[8(n)-³H]-N-ethyl-carboxamidoadenosine ([³H]NECA) binds to the purified protein with a K_D of 155 (144–167) nmol/l and a B_max of 1.85±0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5-N-methylcarb-oxamidoadenosine (CIMECA) and 2-chloro-5-N-ethyl-carboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing K_i values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5-N-ethylcarbox-amidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethylcarbox amidoadenosine (CGS 21680) > R-N⁶-phenylisopropyl-adenosine (R-PIA).Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP 94), tumor rejection antigen precursor (GP96), and some stress related proteins.Abbreviations CGS 21,680 2-[4-(2-carboxyethyl)phenethylamino]-5N-ethylcarboxamidoadenosine - CIA 2-chloroadenosine - CIMECA 2-chloro-5-N-methylcarboxamidoadenosine - CINECA 2-chloro-5-Nethylcarboxamidoadenosine - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - NECA 5-N-ethylcarboxamidoaden-osine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - MECA 5-N-meth-ylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - XAC xanthine amine congener, 8-{4-8[([{(2-aminoethyl)amino}carbonyl]-methyl)oxy]phenyl}-1,3-dipropyl-xanthine Correspondence to: T. Fein at the above adress     

A study of the effects of p,p′-DDE and other related chlorinated hydrocarbons on inhibition of platelet aggregation

A series of chlorinated hydrocarbons of interest in environmental toxicology were investigated concerning their effects on human platelet aggregation. Most potent in inhibiting platelet aggregation were p,p-DDE and Arochlor 1242. Aggregation induced by arachidonic acid (1 mM) was more sensitive to inhibition by p,p-DDE than was aggregation induced by ADP (10 M). The former was completely inhibited by p,p-DDE at a concentration of 1×10^–4M, whereas there was only a 31% inhibition of the latter. Addition of Ca²⁺ (1 mM) blocked the inhibitory effect of p,p-DDE on aggregation induced by both arachidonic acid and ADP. Calmodulin (1 g/ml) blocked the inhibitory effect of p,p-DDE on aggregation induced by arachidonic acid but not that induced by ADP. The calmodulin inhibitory drugs trifluoperazine and calmidazolium had no effect at all or only a weak effect (–14%), respectively, on platelet aggregation. Increasing the concentrations of p,p-DDE and Arochlor 1242 caused a delay in the onset of aggregation induced by the addition of arachidonic acid. The synthesis of thromboxane B₂ and other prostaglandins in platelet membranes was dose-dependently reduced by p,p-DDE. The structurally closely related isomers o,p-DDE and p,p-DDT did not significantly inhibit arachidonic acid-induced platelet aggregation or thromboxane B₂ synthesis. It is concluded that p,p-DDE and Arochlor 1242 inhibited platelet aggregation by inhibiting platelet cyclooxygenase activity.     

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     

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.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Quinidine inhibition of the muscarine receptor-activated K+ channel current in atrial cells of guinea pig

Summary Postsynaptic mechanisms underlying the anticholinergic effects of quinidine were examined in single atrial cells, using the tight-seal whole-cell recording technique. The solution in the glass pipettes contained guanosine-5triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In both cases, acetylcholine (ACh), applied to the bath, induced a specific K⁺ current. In GTP-loaded cells, quinidine in the bath solution depressed the ACh-induced K⁺ current concentration-dependently. Atropine also blocked the K⁺ current. On the other hand, in GTP-S-loaded cells, the ACh-induced current was not blocked by atropine and persisted even when ACh was washed out from the bath, indicating that GTP-S causes uncoupling of the K⁺ channels from the muscarine receptors. Quinidine, however, did depress the increased K⁺ current concentration-dependently. The percent inhibition curves for quinidine to depress the K⁺ current were very similar between GTP-loaded and GTP-S-loaded cells. From these observations, we suggest that direct inhibition of the muscarine receptor-activated K⁺ channel current by quinidine, and not blockade of the muscarine receptor itself, is mainly responsible for the anticholinergic effects of the drug in atrial myocytes. Send offprint requests to Y. Kurachi at the above address     

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     

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.     

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.     

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 Study of 5′-Ester Prodrugs of N6-Cyclopentyladenosine,a Selective A1 Receptor Agonist

Purpose. A series of 5-esters of N⁶-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A₁ agonists. Log P values, stability, affinity, and activity toward human adenosine A₁ receptors were evaluated. Methods. An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A₁ receptor expressed by N⁶-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3-5-cyclic adenosine monophosphate, performing competitive binding assays. Results. All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A₁ receptor of the intact prodrugs. Conclusions. We propose 5-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible canditates for CPA prodrugs.     

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     

[3H]5′-N-ethylcarboxamide adenosine binds to both Ra and Ri adenosine receptors in rat striatum

Summary Adenosine analogs such as 5-N-ethylcarboxamide adenosine and N⁶-cyclohexyladenosine stimulate or inhibit adenosine cyclase activity in preparations of rat striatum depending on the assay conditions. N⁶-cyclohexyladenosine inhibits but does not stimulate adenosine cyclase activity in preparations of hippocampus. These findings suggest that the striatum contains both R _a (stimulatory) and R _i (inhibitory) adenosine receptors while the hippocampus contains only R _i receptors. We have previously shown that [³H]N⁶-cyclohexyladenosine binds to R _i receptors in rat hippocampus (Yeung and Green 1983). Comparisons of the characteristics of [³H]5-N-ethylcarboxamide adenosine and [³H]N⁶-cyclohexyladenosine binding to hippocampus show that [³H]5-N-ethylcarboxamide adenosine also binds to R _i receptors with high affinity. [³H]5-N-ethylcarboxamide adenosine binds to R _i receptors in the striatum and to a second site that is present in striatum but not hippocampus. High affinity binding of both ligands to R _i receptors can be blocked by treatments with N-ethylmaleimide that do not markedly affect [³H]5-N-ethylcarboxamide adenosine binding to the second site in the striatum. The pharmacological characteristics of the second site indicate that it is the R _a adenosine receptor.The abbreviations used are NEM N-ethylmaleimide - Gpp(NH)p 5-guanylylimidodiphosphate - NECA 5-N-ethylcarboxamide adenosine - l-PIA N⁶-(l-phenylisopropyl)adenosine - d-PIA N⁶-(d-phenylisopropyl) adenosine - DPX 1,3-diethyl-8-phenylxanthine     

Effects of p,p′-DDE and some other chlorinated hydrocarbons on the formation of prostaglandins by the avian eggshell gland mucosa

Some structurally related chlorinated hydrocarbons were investigated for their effects on the production of prostaglandins by the eggshell gland mucosa of ducks and domestic fowl. Formation of PGF₂, PGE₂ and TxB₂ by homogenates of domestic fowl eggshell gland mucosa was significantly inhibited by in vitro addition of p,p-DDE, Arochlor 1242 and, to a lesser extent, Arochlor 1260, but not by p,p-DDT and o,p-DDE. Comparatively, in duck eggshell gland mucosa homogenates, synthesis of the same prostaglandins was somewhat more sensitive to inhibition by 5 M p,p-DDE added in vitro. Eggshell gland mucosa synthesized significantly more PGF₂, PGE₂ and TxB₂ than did the mucosa of the magnum and isthmus regions of the oviduct. Duck eggshell gland mucosa homogenates synthesized significantly more prostaglandins than similar homogenates from the domestic fowl, and, considering the former synthesis of PGF₂ was significantly higher when ducks were slaughtered at 08:00 than at 16:00 hours. In ducks, dietary administration of 40 ppm, p,p-DDE for 45 days resulted in 21% eggshell thinning compared to the contemporary control values. This treatment also resulted in notable effects in homogenates of the eggshell gland mucosa, as compared to controls: Ca²⁺ uptake was reduced by 43%, synthesis of PGF₂, PGE₂ and TxB₂ was reduced by 26%, 38% and 53%, respectively; the Ca content was increased to 145%. The role of p,p-DDE in inhibiting prostaglandin formation in the eggshell gland is discussed as a mechanism of the eggshell thinning action of this chlorinated hydrocarbon.     

Subtype specificity of γ-aminobutyric acid type A receptor antagonism by clozapine

Clozapine, an atypical neuroleptic, functionally antagonizes the -aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, -aminobutyric acid type A (GABA_A) receptor, in brain vesicles. GABA_A antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA_A antagonist 2-(3-carboxy-2,3-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [³⁵S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [³H]muscimol and [³H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [³⁵S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant 622 receptors, the predominant 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 M. In contrast, recombinant 122 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant 622 and 632 receptors resulted in clozapine-insensitive receptors, whereas 612 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in 1x2 receptors decreased in the order of 112>122>132. The results indicate that clozapine antagonizes the function of most GABA_A receptor subtypes, and that the interaction is determined by the interaction of the and subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment.