Low-affinity interactions of BODIPY-FL-GTPγS and BODIPY-FL-GppNHp with G<Subscript>i</Subscript>- and G<Subscript>s</Subscript>-proteins

BODIPY-FL-guanosine 5'-[-thio]triphosphate (B-GTPS) and BODIPY-FL-guanosine 5'-[,-imido]triphosphate (B-GppNHp) induce fluorescence changes upon binding to purified G_s/G_i-proteins and were suggested to serve as probes for monitoring receptor-mediated G-protein activation. However, B-GTPS and B-GppNHp bound to receptor-G_s/G_i fusion proteins expressed in Sf9 cell membranes with 1,100- to 5,600-fold- and 17- to 55-fold lower affinity than GTPS and GppNHp, respectively. The affinity of B-GTPS/B-GppNHp for G_s/G_i-proteins was considerably lower than the affinity of N-methylanthraniloyl (MANT)-substituted GTP analogs for G_s/G_i-proteins. B-GTPS/B-GppNHp were much less potent than GTPS/GppNHp at regulating adenylyl cyclase (AC) via G_s- and G_i-proteins. B-GTPS/B-GppNHp were similarly efficient as GTPS/GppNHp at activating G_i, but less efficient at activating G_s. In contrast to MANT-GTPS/MANT-GppNHp, B-GTPS/B-GppNHp were inefficient at directly inhibiting AC. In conclusion, the bulky BODIPY group strongly reduces the affinity of GTPS/GppNHp for G-proteins, limiting the use of B-GTPS/B-GppNHp as fluorescence probes.Abbreviations AC Adenylyl cyclase - ₂AR ₂-Adrenoceptor - ₂AR-G_olf Fusion protein consisting of the ₂-adrenoceptor and G_olf - ₂AR-G_sL Fusion protein consisting of the ₂-adrenoceptor and the long splice variant of G_s - ₂AR-G_sS Fusion protein consisting of the ₂-adrenoceptor and the short splice variant of G_s - B-GppNHp BODIPY-FL-guanosine 5'-[,-imido]triphosphate - B-GTPS BODIPY-FL-guanosine 5'-[-thio]triphosphate - FPR-G_i1,2,3 Fusion protein consisting of the formyl peptide receptor and G_i1, G_i2 or G_i3 - G Unspecified G-protein -subunit - G_i Inhibitory G-protein of adenylyl cyclase - G_olf Olfactory G-protein that activates adenylyl cyclase - G_s Stimulatory G-protein of adenylyl cyclase - G_sL Long splice variant of the stimulatory G-protein of adenylyl cyclase, G_s - G_sS Short splice variant of the stimulatory G-protein of adenylyl cyclase, G_s - GppNHp Guanosine 5'-[,-imido]triphosphate - GTPS Guanosine 5'-[-thio]triphosphate - M-GppNHp 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[,-imido]triphosphate (MANT-GppNHp) - M-GTPS 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[-thio]triphosphate (MANT-GTPS)     

Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, β-carbolines,and phenobarbitone

Summary The potent benzodiazepine receptor ligands -carboline-3-carboxylic acid ethyl ester (-CCM) and the corresponding methylester (-CCM) administered i.v. depressed segmental dorsal root potentials in spinal cats, reversed the prolongation of dorsal root potentials by phenobarbitone, and abolished the depression of a motor performance task induced by phenobarbitone in mice; -CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of -carbolines reflect a depression of GABAergic synaptic transmission and, thus, are diametrically opposed to the enhancing action of benzodiazepine tranquilizers. The specific benzodiazepine antagonist, Ro 15-1788, while not affecting dorsal root potentials, hippocampal population spikes or phenobarbitone-induced motor performance depression, abolished the effects of -CCE on the three parameters and similar effects of -CCM on the spinal cord and motor performance.A three-state model of the benzodiazepine receptor is proposed in which benzodiazepine tranquilizers act as agonists enhancing the function of the benzodiazepine receptor as a coupling unit between GABA receptor and chloride channel, -carbolines act as inverse agonists reducing this coupling function, and Ro 15-1788 represents a competitive antagonist blocking both the enhancing effect of agonists and the depressant effect of inverse agonists on GABAergic synaptic transmission.     

Incorporation of the biomarker concept in ecotoxicology calls for a redefinition of terms

Biomarkers have received a lot of attention but the biomarker concept has not yet been defined properly. As a consequence, various interpretations of the term biomarker exist, some of which overlap with well-established ecotoxicological concepts. To allow incorporation of the biomarker concept within the framework of modern ecotoxicology, a clarification of terms is needed. In this paper, definitions are presented for the terms biomarker, bioindicator and ecological indicator, which do not overlap and are linked to different levels of biological organization. The use of these concepts for ecological effects assessment is discussed.     

Distinct distribution of opioid receptor types in rat lumbar spinal cord

Summary The distribution of opiate binding sites was studied in sections of rat lumbar spinal cord under conditions selective for , and receptors. While the levels of binding sites were highest in the substantia gelatinosa, elevated levels were also observed in laminae III, IV, V and VIII. In contrast, binding was notable only in lamina I. The levels of typical sites were low, and were concentrated in the substantia gelatinosa. An additional, atypical site was detected using ³H-diprenorphine in the presence of , and receptor blocking agents, and this site was also concentrated in the substantia gelatinosa. Send offprint requests to B. J. Morris at the above address     

Interaction of adenine nucleotides,UTP and suramin in mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect of ATP

Summary Effects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. ,-Methylene-ATP, ATPS, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 mol/l). Prolonged incubation with ,-methylene-ATP (concentration increased stepwise from 0 to 15 mol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10–300 mol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for ,-methylene-ATP, ATPS, UTP and lower concentrations of ATP (0.1–1 ol/l) to the right. The pA₂-values of suramin were 5.2 against ,-methylene-ATP, 4.8 against ATPyS, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P_2X-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P₂-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P_2X-receptor.Send offprint requests to I. von Kügelgen at the above address     

Über die Wirkung von α-Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen

Zusammenfassung 1. Versuche über die Wirkung von -Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen haben ergeben, daß Noradrenalin im Herzen, sowie Dopamin und Noradrenalin im Gehirn durch äquimolare Mengen von -Methyl-Noradrenalin bzw. -Methyl-Dopamin ersetzt werden.2. Die Adrenalinverarmung der Nebennieren nach Vorbehandlung mit -Methyl-Dopa wird demgegenüber nur zu einem geringen Teil durch die methylierten Brenzcatechinamine -Methyl-Noradrenalin und -Methyl-Dopamin ausgeglichen. Ein weiteres Brenzcatechinamin konnte nicht nachgewiesen werden, so daß -Methyl-Adrenalin wahrscheinlich nicht gebildet wird.3. Im Meerschweinchenherzen gespeichertes -Methyl-Noradrenalin wird durch Reserpin in vivo schlechter freigesetzt als Noradrenalin.4. Das in isolierten Herzgranula (Sediment 100 000·g) gespeicherte -Methyl-Noradrenalin wird durch Segontin bei der Inkubation in vitro ebenfall schlechter freigesetzt als Noradrenalin aus normalen Herzgranula.5. Der Mechanismus der -Methyl-Dopa-Wirkung wird diskutiert.6. Es wird eine fluorimetrische Methode zur Bestimmung von -Methyl-Noradrenalin angegeben.

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Summary 1. The catecholamine content of guinea pig organs after administration of -methyldopa has been determined. The results show that the loss of noradrenaline from heart and that of noradrenaline and dopamine from brain is compensated by a stoichiometric uptake of -methylnoradrenaline and -methyldopamine respectively.2. On the contrary the loss of adrenaline from the suprarenal medulla induced by pretreatment with -methyldopa is not completely restored by the -methylated amines, -methyldopamine and -methylnoradrenaline. -methyladrenaline could not be detected even by paperchromatography.3. The release of the stored -methylnoradrenaline from guinea pig heart by reserpine is very small in comparison with that of noradrenaline.4. Furthermore only small amounts of the stored -methyl-noradrenaline are released from isolated granules of the guinea pig heart after incubation in vitro with segontin. Whereas the same amount of segontin depleted completely the noradrenaline content of the granules isolated from normal hearts.5. The mechanism of action of -methyldopa is discussed.6. A method for the fluorimetric determination of -methylnoradrenaline is described. It is at first separated from -methyldopamine, dopamine and histamine by column chromatography, condensed with o-phthaldialdehyd and the obtained fluorescense is measured by using an Aminco-Bowman spectrophotofluorimeter.

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Mit 6 Textabbildungen

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Herrn Dr. H. Blaschko zum 65. Geburtstag gewidmet.

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Über einen Teil der Ergebnisse wurde auf der Frühjahrstagung der Deutschen Pharmakologischen Ges. in Mainz, April 1964, berichtet. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 297 (1964).

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

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).     

Two relaxation-mediating P2-purinoceptors in guinea-pig taenia caeci

2-Methylthio ATP, noradrenaline, adenosine 5-O-(2-thiodiphosphate) (ADP\S), ,\-methylene ATP, ATP and adenosine caused relaxation of guinea-pig isolated taenia caeci precontracted by carbachol, with potency decreasing in the order indicated. 4,4-Diisothiocyanatostilbene-2,2-disulphonate (DIDS) 10, 32 and 100 M shifted the concentration-response curve of ,\-methylene ATP increasingly to the right, by the factor 141 at DIDS 100 M. Concentration-response curves of the other agonists were not shifted to the right by DIDS 100 M, except for the curve of ADP\S which was shifted by the factor 2.4. The relaxation produced by 2-methylthio ATP faded rapidly. When the fade was complete, further addition of 2-methylthio ATP or ATP did not elicit relaxation, whereas the relaxant effect of , \-methylene ATP was unchanged. The results indicate that there are at least two relaxation-mediating P₂-purinoceptors in guinea-pig taenia caeci, the P_2Y-purinoceptor which is relatively insensitive to DIDS and a distinct receptor for ,\-methylene ATP which is very sensitive to DIDS.     

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

Summary Four groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (K _I, nM) were determined against the binding of the -ligand, [³H]-[d-Ala ² ,MePhe ⁴ , Gly-ol⁵]enkephalin, of the -ligand, [³H]-[d-Ala ² ,d-Leu ⁵]enkephalin and of the -ligand, [³H]-(±)-ethylketazocine after suppression of - and -binding by 100 nM of the unlabelled -ligand and 100 nM of the unlabelled -ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to -binding but phenazocine and particularly etorphine had also high affinities to the - and -binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, -binding and -binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the -binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective -antagonist, Mr 2266 having high affinities to the - and -binding sites and diprenorphine having considerable affinities to the -, - and -binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the - and -binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.     

Isoprenaline-induced increase in mRNA levels of inhibitory G-protein α-subunits in rat heart

Summary Long-term -adrenergic stimulation has been shown to desensitize the -adrenoceptor/adenylyl cyclase signalling pathway at both the receptor and the G-protein level. To further elucidate the cellular mechanism of G-protein regulation we investigated the influence of prolonged infusion of isoprenaline (2.4 mg/kg·d) on myocardial mRNA levels of different G-protein -subunits in rats. For comparison rats were treated with triiodothyronine (T₃; 0.5 mg/kg·d) which induces cardiac hypertrophy like isoprenaline but has different effects on the adenylyl cyclase system. Isoprenaline- and T₃-treated animals developed an increase in heart/body weight ratio of 41±3% and 27±4%, respectively (P<0.05). Isoprenaline increased myocardial total RNA concentration by 39±6% (P<0.05). Hybridization with ³²P-labeled rat cDNAs demonstrated an expression rank order of G_s-mRNA>G_i-2-mRNA>G_i–3-mRNA and no detectable expression of G_i–1-mRNA in rat myocardium. mRNA levels of G_s G_i–2 and G_i–3 were 36.9±1.28, 10.7±1.07 and 3.7±0.19 pg/g total RNA, respectively. Isoprenaline increased G_i–2 ^– and G_i–3-mRNA concentrations per g total RNA by 49±18% and 27±710, respectively (P<0.05). This effect was abolished by simultaneously administered propranolol (9.9 mg/kg·d), indicating a,-adrenoceptor-mediated mechanism. In contrast, T₃-induced cardiac hypertrophy was not accompanied by changes in G_i-mRNA expression. G_sa-mRNA levels were unaffected by either treatment.In conclusion, long-term stimulation with isoprenaline in vivo induces a -adrenoceptor-mediated increase in myocardial G_i–2 ^– and G_i–3-mRNA without affecting G_s-mRNA. These results suggest that similar increases in myocardial G_i–2-mRNA in end-stage human heart failure may be at least partly explained by increased -adrenergic stimulation due to increased sympathetic activity.Parts of this work were presented at the wintermeeting of the Deutsche Gesellschaft fur Pharmakologie und Toxikologie in Hannover, 1990 (Eschenhagen et al.), Naunyn-Schmiedebergs Arch Pharmacol 342 (Suppl):R8. The work was supported by the Deutsche Forschungsgemcinschaft Send offprint requests to: T. Eschenhagen at the above address     

The role of neuronal uptake atα- andβ-adrenoceptor sites in subcutaneous adipose tissue

Summary Intravascular noradrenaline infusion may cause vasodilatation or vasoconstriction in subcutaneous adipose tissue, whereas sympathetic nerve activity causes vasoconstriction only. This discrepancy may be due to a differential distribution of - and -adrenoceptors in relation to adrenergic nerve terminals in the adipose tissue vessels. In order to test this hypothesis the extent of prejunctional supersensitivity to noradrenaline was studied after blockade the neuronal uptake of noradrenaline with cocaine.In the autoperfused, isolated inguinal canine adipose tissue pretreatment with cocaine (200–600 g close i.a.) increased lipolysis following sympathetic nerve stimulation or close i.a. injection of noradrenaline. Cocaine also potentiated the vasoconstriction induced by nerve stimulation (1–3 Hz) or intra-arterial noradrenaline (0.2–2 nmoles) as well as the vasodilatation induced by sympathetic nerve stimulation (1–3 Hz) after -receptor blockade. However, the vasodilatation following close i.a. injection of noradrenaline after -receptor blockade was not changed by cocaine.The results indicate that the functionally important vascular -adrenoceptors in adipose tissue are in close contact with adrenergic nerve terminals, whereas most vascular -adrenoceptors seem to be unrelated to the nerve terminals. Thus, the -adrenoceptors in the adipose tissue vessels may be classified as innervated receptors, in contrast to the vascular -adrenoceptors which may be more acessible to circulating catecholamines and may be classified as humoral receptors. Furthermore at least some of the -receptors on the adipocytes seem to be located close to sympathetic nerve terminals.     

The in vitro/in vivo comparative metabolism of 4-aminobiphenyl using isolated hepatocytes

The metabolism of 4-aminobiphenyl by isolated hepatocytes from various species was compared with urinary metabolite profiles in the same species. Radioactive compounds in concentrates of ether extracts from hepatocytes or urine following hydrolysis were analysed by TLC and reversed phase HPLC in conjunction with radioactivity monitoring and synthetic standards.The major metabolites from hepatocytes and in urine were 4-acetamidobiphenyl, 3-hydroxy-4-aminobiphenyl 4-hydroxy-4-aminobiphenyl and 4-hydroxy-4-acetamidobiphenyl. Oxidation of the amine nitrogen gave hydroxylamino, nitroso and nitro compounds. Minor metabolites were 2-hydroxy amine and amide, the hydroxamic acid and the oxamic acid. The urinary metabolite profiles correlated well with those from hepatocytes for each species.Abbreviations Used 4-ABP 4-aminobiphenyl - AA 4-acetamidobiphenyl - A3-OH 3-hydroxy-4-aminobiphenyl - A4-OH 4-hydroxy-4-aminobiphenyl - A2-OH 2-hydroxy-4-aminobiphenyl - AA4-OH 4-hydroxy-4-acetamidobiphenyl - AA3-OH 3-hydroxy-4-acetamidobiphenyl - AA2-OH 2-hydroxy-4-acetamidobiphenyl - AAN-OH N-hydroxy-4-acetamidobiphenyl - NBP 4-nitrobiphenyl - AN-OH 4-hydroxylaminobiphenyl - NOBP 4-nitrosobiphenyl Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Plasma insulin levels and β-adrenoceptor antagonists

Summary The effect of -adrenoceptor antagonists on the intravenous glucose tolerance test was investigated in conscious dogs. dl-Celiprolol (cardioselective with ISA=intrinsic sympathomimetic activity) 200 and 1000 g/kg i.v., dl-metoprolol (cardio-selective without ISA) 200 and 1000 g/kg i.v., dl-pindolol (non-selective with ISA) 5 and 25 g i.v. and l-bupranolol (non-selective without ISA) 10 and 50 g/kg i.v. were used in the study. The influence of -adrenoceptor antagonists on the plasma glucose and immunoreactive insulin following the intravenous glucose tolerance test were evaluated by calculating the respective areas under the plasma curve.The present investigtion clearly demonstrates the marked difference between the various -adrenoceptor antagonists on heart rate and, especially on metabolic parameters. dl-Metoprolol, a -adrenoceptor antagonist with cardioselectivity and without ISA can be assumed not to alter plasma insulin level and glucose assimilation. l-Bupranolol, a non-selective -adrenoceptor antagonist without ISA reduces plasma insulin level and probably enhances peripheral glucose uptake, resulting in an unchanged glucose tolerance. dl-Celiprolol or dl-pindolol, -adrenoceptor antagonists with ISA, but cardioselective or non-selective enhance both, basal insulin level and insulin level after glucose stimulation but must be assumed to decrease peripheral glucose uptake since here too glucose tolerance was unchanged.     

Pharmacokinetics of canrenone and metabolites after base hydrolysis following single and multiple dose oral administration of spironolactone

Summary The pharmacokinetics of canrenone and total metabolites after base hydrolysis was studied in eight young volunteers following single and multiple dose oral administration of spironolactone. The plasma levels of canrenone and total metabolites were fitted to a two-compartment open model with a first-order absorption process. From our eight normal subjects studied, the harmonic mean of the distributive half-life (t_1/2) of canrenone was found to be 1.66 h, and the harmonic mean of the terminal elimination half-life (t_1/2) to be 22.6 h. Harmonic means of the distributive and elimination half-lives of total metabolites after base hydrolysis were 2.48 h and 28.8 h respectively. The accumulation ratio of canrenone was 2.53, whereas that of total metabolites was 1.89. Despite the fact that spironolactone has been shown to induce hepatic metabolism of other drugs, no evidence of autoinduction was noted in the present study, as plasma levels of canrenone and total metabolites were found to obey a linear two-compartment model with reproducible absorption and disposition after single and multiple doses.     

Is there a need for more precise definitions of bioavailability?

Summary After evaluation of the present definitions in a set of particular cases, it was agreed that there was no need for more precise definitions and that the current ones were adequate in the majority of cases. However, it was felt that the present definitions might be improved, in particular in view of the existence of non-systemically acting drugs and future targeted drugs. Thus, the FDA definition might be modified as follows: Bioavailability means the rate and extent to which the active drug ingredient or therapeutic moiety from a drug product becomes available at the site of drug action or in a biological medium believed to reflect accessibility to a site of action.     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Failure of “antigastrin” (SC-15 396) to inhibit gastric acid and pepsin secretion in anaesthetized gastric fistula cats

Summary 1. The effect of antigastrin (SC-15 396) on gastric acid and pepsin secretion produced by the gastrin-analogue tetrapeptide amide Try. Met. Asp. Phe-NH₂ and by electrical stimulation of the vagus was investigated in anaesthetized gastric fistula cats.2. Antigastrin failed to inhibit both acid and pepsin response stimulated by either the tetrapeptide or vagus excitation.3. It was concluded that the ineffectiveness of antigastrin in cats is due to a species difference between rats and dogs on the one hand and cats on the other, and that antigastrin is not a specific gastrin antagonist.Supported by the Deutsche Forschungsgemeinschaft and by the Alfred Teufel-Stiftung.     

Interaction of Human IgE with Soluble Forms of IgE High Affinity Receptors

Purpose. Interaction of human IgE with its high affinity receptor (FcRI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and FcRI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE FcRI chain, the extracellular portion of IgE high affinity receptor (sFcRI) was compared with the binding of IgE and IgE immunoadhesin (FcRI-IgG). Methods. The interaction was characterized by analytical ultracentrifugation, size exclusion chromatography, light scattering and ELISA. Results. We show that the sFcRI is only able to bind to one IgE, while the immunoadhesin can bind to two IgE. The interaction between IgE and FcRI is very strong. Both forms of soluble receptors have similar intrinsic binding affinity with IgE. Conclusions. Both soluble receptors (FcRI-IgG and sFcRI) can block the binding of IgE to its high affinity receptors on cell surface. The FcRI-IgG is a better IgE binding protein than sFcRI at physiological relevant conditions. A humanized anti-IgE monoclonal antibody, rhuMAb E25 that also can block the binding of IgE to its high affinity receptors appears to bind to IgE at slightly different regions or in a different manner as the soluble forms of IgE receptors.     

Effects of the phenethylamine derivatives,BL-3912, fenfluramine,and Sch-12679, in rats trained with LSD as a discriminative stimulus

Six rats were trained to discriminate the effects of LSD (100 g/kg) and saline in a two-lever choice task. They were then tested with each of three phenethylamine derivatives, BL-3912 (2,5-dimethoxy-4-methyl--ethyl-phenethylamine), fenfluramine (N-ethyl--methyl-m-(trifluoro-methyl)phenethylamine), and Sch-12679 (N-methyl-1-phenyl-7,8-dimethoxy-2,3,4,5-tetra-hydro-3-benzazepine maleate). Fenfluramine and Sch-12679 yielded intermediate results, i.e., responding was not fully appropriate for either training condition while BL-3912 substituted completely for LSD. The LSD-like effects of each of the drugs were antagonized by pretreatment with BC-105, a serotonergic antagonist known to block the stimulus effects of indole and phenethylamine hallucinogens. The present data together with consideration of the known clinical effects of BL-3912, fenfluramine, and Sch-12679 are consistent with the following conclusions: (1) a variety of drugs may substitute in whole or in part for LSD in LSD-trained rats, and (2) even complete substitution of a drug for LSD in the rat is not necessarily associated with the production by that drug of hallucinations in man.