α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Pharmacokinetics of digoxin and its 4‴-acetyl-and methylderivates in the rat

Summary The kinetics of absorption, of changes in blood concentration, and of biliary excretion after the i.v. and i.d. administration of 40 Ci each, of digoxin, 4-acetyldigoxin and 4-methyldigoxin were studied in biliary fistula rats. The highest blood concentrations were found after the i.v. administration of 4-methyldigoxin, which decline with a half life time of 10 h, compared with 5.6 and 4.5 h for 4-acetyldigoxin and digoxin respectively. 71%, 55% and 17% of the dose were excreted in the bile within 12 h after the i.v. administration of digoxin, 4-acetyldigoxin and 4-methyldigoxin. The blood concentrations observed after the i.d. administration of digoxin and 4-acetyldigoxin show almost identical pharmacokinetics with respect to height and elimination velocity (half life 7.0 h for digoxin and 7.5 h for 4-acetyldigoxin). In contrast, following the i.d.administration of 4-methyldigoxin, blood concentrations, which were twice as high, were observed and declined with the same half life as after the i.v. administration.Determination of the disappearance rates of these glycosides from the intestinal lumen reveals a biphasic course of absorption. A first phase, with k values of 0.4, 0.5, 1.2 for digoxin, 4-acetyldigoxin and 4-methyldigoxin respectively is followed by a second phase with k values of 0.04, 0.04, 0.001 for digoxin, 4-acetyldigoxin and 4-methyldigoxin. Thus, 4-methyldigoxin is almost completely absorbed within the first two hours, while digoxin and 4-acetyldigoxin continue to be absorbed during the following hours. The absorption velocity of digoxin from the ileum was found to be one half of that seen in the duodenum. But this slow absorption, as well, follows a biphasic course.The data indicate that 4-methyldigoxin is absorbed at a distinctly higher rate than 4-acetyldigoxin and digoxin. Acetylation in 4 position evidently provides no important advantage with respect to absorption. While this study allows the determination of absorption and excretion velocities, no account of absorption quotes is given.     

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.     

Evidence for two separate vasoconstriction-mediating nucleotide receptors,both distinct from the P2X-receptor,in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

Summary Uridine 5-triphosphate- (UTP-) and adenosine 5-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5-O-(3-thio)triphosphate (ATPS), and ,-imido adenosine 5-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, , -methylene-ATP and ,-methylene-ATP up to 10^–3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP UMP = CTP; that of the purine nucleotides was: ATPS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = , -methylene-ATP = ,-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to ,-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10^–3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10^–3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10^–3 to 2 × 10^–3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P₂-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P₂-receptor which is distinct from the known P₂-subtypes.Send offprint requests to I. v. Kügelgen at the above address     

The significance ofβ-adrenoceptor down regulation in the desipramine action in the forced swimming test

Summary The present studies were undertaken to clarify whether central-adrenoceptor down regulation is responsible for the greater effect of chronic treatment with desipramine (DMI) compared with acute treatment in the forced swimming test in rats. Repetitive administration of DMI activated the rat behaviour pattern and consequently reduced the duration of immobility. The degree of activation depended on the length of treatment, i.e. no effect when given in a single dose, moderate effect when given subchronically (3 doses) and marked activation after chronic (31 doses) treatment. Chronic treatment with DMI also produced a decrease in³H-dihydroalprenolol (³H-DHA) binding site in the cerebral cortex. Acute stimulation of brain-adrenoceptors by intracerebroventricular (i.c.v.) isoprenaline significantly, though partially, attenuated the behavioural effect of chronic DMI by ₁-adrenoceptor-related mechanisms. Similarly, chronic i.c.v. co-administration of atenolol or practolol, ₁-adrenoceptor antagonists, together with DMI attenuated both-adrenoceptor down regulation and the behavioural activation by chronic DMI. On the other hand, chronic i.c.v administration of isoprenaline, supposedly leading to down regulation of-adrenoceptors, facilitated the activating behavioural effect of DMI, as a single dose became effective. Changes, however, in³H-DHA binding parameters in the cerebral cortex were not observed after chronic isoprenaline. These results suggest that down regulation of-adrenoceptors in brain is reponsible, at least in part, for the marked activatory effect of chronic DMI in the forced swimming test, possibly by reducing an inhibitory function of ₁-adrenoceptor mediated mechanisms.     

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.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address     

Behavioural and anticonvulsant effects of Ca2+ channel toxins in DBA/2 mice

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice.-Conotoxin MVIIC (0.1, 0.3 g ICV/mouse) and-agatoxin IVA (0.1, 0.3, 1 g ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED₅₀ values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04–0.36) g ICV and 0.09 (0.05–0.15) g ICV, respectively and against tonic seizures 0.07 (0.03–0.16) g ICV and 0.08 (0.04–0.13) g ICV, respectively). The N-type calcium channel antagonists-conotoxin GVIA and-conotoxin MVIIA were also tested in this model.-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 g ICV prevented tonic seizures in 60% of the animals; 10 g ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 g ICV. Both-conotoxin GVIA and-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 g ICV, whereas-conotoxin MVIIC and-agatoxin IVA did not produce shaking at any of the doses examined. Finally,-conotoxin GI (0.01–1 g ICV) and-conotoxin SI (0.3–30 g ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of-conotoxin MVIIC and-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.     

Pre- and postganglionic stimulation-induced noradrenaline overflow is markedly facilitated by a prejunctional β2-adrenoceptor-mediated control mechanism in the pithed rat

The aim of the study was to further explore the prejunctional -adrenoceptor-mediated control mechanism of noradrenaline release from sympathetic nerves in response to preganglionic nerve stimulation (PNS) and local nerve stimulation of the portal vein, respectively, in the pithed rat.Baseline values as well as the increments of mean arterial blood pressure (-BP), heart rate (-HR) and plasma noradrenaline levels (-NA) in response to four PNS episodes (0.8 Hz, 3 ms, 75 V for 45 s at 20 min intervals), respectively, were evaluated. Fenoterol administration (0.25 mg/kg, i.v.) reduced significantly the basal blood pressure but did not alter d-BP in response to PNS. Basal heart rate markedly increased after fenoterol without any further change in heart rate induced by PNS.The ₁-selective antagonist CGP 20712A attenuated -BP in response to PNS and prevented the fenoterol-induced increase in basal heart rate. The ₂-selective antagonist ICI 118,551 per se did not change the blood pressure and heart rate values, but antagonized the fenoterol-induced decrease in basal blood pressure.Fenoterol enhanced plasma -NA in response to PNS by 105% in comparison to the corresponding control value. This effect of fenoterol could be blocked by pretreatment with ICI 118,551 but not with CGP 20712A (a selective ₁-adrenoceptor antagonist) which per se did not significantly change plasma -NA.Repeated local stimulation of the portal vein (S1–S 3, 2 Hz, 3 ms, 10 mA, for 120 s at 30 min intervals) increased portal plasma noradrenaline without changing mean blood pressure and heart rate in pithed rats. Fenoterol enhanced the increase in portal-vein plasma noradrenaline evoked by nerve stimulation by 110%. Pretreatment with ICI 118,551 antagonized this effect of fenoterol, but had per se no effect on the portal vein nerve stimulation-evoked increase in portal plasma noradrenaline.It is concluded that the increase in plasma noradrenaline evoked both by pre- and postganglionic nerve stimulation can be markedly enhanced by activation of a facilitatory prejunctional ₂-adrenoceptor control mechanism. Correspondence to: V.I. Tarizzo     

Desensitization of kitten atria to chronotropic,inotropic and adenylyl cyclase stimulating effects of (-)isoprenaline

Summary Desensitization of kitten atria with 30 M (-)isoprenaline resulted in a 6-fold and 15-fold increase in the EC₅₀'s of (-)isoprenaline for its positive chronotropic effects (sinus pacemakers) and positive inotropic effects (left atria), respectively, but only in a 2-fold increase of the EC₅₀ of (-)isoprenaline for adenylyl cyclase stimulation in membrane particles from atria. However, maximum cyclase stimulation by (-)isoprenaline was decreased to 1/2 in membranes from (-)isoprenaline-treated atria, whereas maximum increases in rate of sinus pacemakers and force of left atria were unchanged and reduced by 15%, respectively. The high affinity -adrenoceptor blocker (-)bupranolol antagonized the adenylyl cyclase stimulation by (-)isoprenaline to similar extent in membranes from (-)isoprenaline and untreated atria, suggesting that the apparent affinity of -adrenoceptors for ligands is unchanged by desensitization. The evidence is compatible with the concept that desensitization is associated with decreased availability of receptors and with the view that near maximal positive chronotropic effects of catecholamines may be caused by only threshold increases in membrane adenylyl cyclase activity.     

Preparation of Heptakis(2,6-di-O-ethyl)-β-cyclodextrin and Its Nuclear Magnetic Resonance Spectroscopic Characterization

Heptakis(2,6-di-O-ethyl)--cyclodextrin (DE--CyD) was prepared and its ¹H and ¹³C nuclear magnetic resonance (NMR) signals in DMSO-d₆ were unequivocally assigned by two-dimensional COSY and ROESY. The results on ¹H coupling constants indicated that all ethylated glucose units are in a ⁴C₁ chair conformation. The average spin-lattice relaxation times (T ₁) of ring carbons of DE--CyD were only slightly shorter, and their standard deviations from the mean T _l value were larger, than those of -cyclodextrin (-CyD) and heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), suggesting the presence of slightly irregular internal motion in the ethylated glucose units. The temperature dependence of chemical shift of DE--CyD in DMSO-d₆ suggested that the C3 hydroxyl protons may participate as proton donor in the intramolecular hydrogen bond to the C2 ethoxyl groups of neighboring glucose, and the intramolecular hydrogen bond of DE- and DM--CyDs is much stronger than that of -CyD, suggesting the stable macrocyclic ring structure of DE--CyD.     

Disposition Kinetics of α-Tocopherol in Apolipoprotein B Knockout Mice

Purpose. We examined the effects of apolipoprotein B (apoB) on the disposition kinetics of -tocopherol by using apoB knockout mice. Methods. The concentrations of -tocopherol in plasma and tissues were measured by gas chromatography-mass spectrometry. Results. In apob (–/–) mice, the endogenous levels of -tocopherol in plasma and tissues (except liver) were significantly lower, and the liver concentration was significantly higher than those in wild-type mice. After single i.v. administration of -tocopherol (25 mg/kg), the area under the plasma concentration-time curve (AUC) and the distribution volume at steady state were significantly decreased, whereas the total clearance of -tocopherol was significantly increased in apob (–/–) vs. wild-type mice. -Tocopherol was highly distributed to the liver, compared with other tissues. After an oral administration of -tocopherol (100 mg/kg), the intestinal absorption of -tocopherol was very low in apoB knockout mice, as the value of AUC_0-32h for apob (–/–) mice (17.7 ± 8.3 g h/mL) was significantly less than that for apob (+/+) wild-type mice (96.5 ± 15.8 g h/mL, mean ± SD of five experiments, p < 0.01). The biliary excretion of -tocopherol was significantly greater in apob (+/–) mice than in apob (+/+) mice. Conclusions. These results show that apoB plays a role in hepatic secretion and intestinal absorption of -tocopherol.     

Liver growth and mixed-function oxidase activity: Dose-dependent stimulatory and inhibitory effects of α-hexachlorocyclohexane

Summary -hexachlorocyclohexane (-HCH) elicits liver growth and stimulation of hepatic microsomal mixed-function oxidase. In the present study the extent of these changes was determined in rats 2, 4 and 6 days after treatment with doses of -HCH ranging from 1 to 600 mg/kg. Above the respective threshold doses liver mass, liver DNA, and the rate of aminopyrine demethylation increased in proportion to the log dose. Threshold doses were calculated to be 30 mg/kg for the increase of liver weight and DNA, and 5 mg/kg for the stimulation of aminopyrine demethylation.Liver mass and liver DNA continued to increase up to the highest dose used; in contrast, the rate of aminopyrine demethylation declined at doses exceeding 200 mg/kg. This decline seems to be due to inhibition by -HCH retained in the microsomal fraction: -HCH is a potent, apparently competitive inhibitor of aminopyrine demethylation, and gaschromatographic determinations revealed that the amount of -HCH retained in the microsomes is sufficient to produce considerable inhibition of the enzymatic reaction.Abbreviations -HCH -1,2,3,4,5,6-hexachlorocyclohexane, -benzene hexachloride - EL 241 [,-bis(p-chlorophenyl)-3-pyridinemethanol] - RLW relative liver weight = liver weight in percent of body weight     

Two distinct adrenoceptor-biophases in the vasculature: One for α-and the other for β-agonists

Summary Strips of two canine vessels with different patterns of sympathetic innervation were used: the mesenteric artery which has an adventitio-medial plexus and the saphenous vein in which nerve terminals are distributed throughout the media.The pD₂ of (–)-adrenaline for -and -adrenoceptors was determined for both vessels (in the presence of 0.5 M propranolol and 7 M phentolamine, respectively; in the latter case the strips were contracted by 0.28 M prostaglandin F2) and found to be very similar: 6.96 and 7.01 in the saphenous vein and 6.77 and 6.91 in the mesenteric artery, respectively. The similarity of pD₂ for adrenaline acting on -and -adrenoceptors in both preparations allowed us to compare the effect of inhibition of neuronal uptake by 12 M cocaine with that of inhibition of COMT by 50 M dihydroxy-2-methyl propiophenone (U-0521) on: a) the potency of adrenaline (noradrenaline was used in the saphenous vein only) acting on -and -adrenoceptors and b) the time required by the strips to recover 50% in oil (t ₅₀) after contractions or relaxations caused by 0.23 M adrenaline. In the saphenous vein cocaine increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (2.8 times vs. no increase for adrenaline; 7.1 vs. 1.9 times for noradrenaline) and U-0521 increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (4.1 vs. 2.6 times for adrenaline; 1.8 times vs. no increase for noradrenaline); regarding the termination of action of adrenaline, cocaine prolonged the t ₅₀ 1.6 times after contraction and did not change it after relaxation, whereas U-0521 prolonged the t ₅₀ 6.9 times after relaxation and only 1.8 times after contraction.In the mesenteric artery only sensitivity experiments were done. Cocaine increased the potency of adrenaline by a factor of 2.1 for the -effects while there was no influence on its potency with regard to its -effects, and U-0521 increased the potency of adrenaline for the -effects more than for -effects (3.9 vs. 1.8 times, respectively).These results show that there are two different biophases for sympathomimetic agonists in the vasculature: one for -adrenoceptors which is more under the influence of the neuronal uptake and one for -adrenoceptors which is more under the influence of COMT activity. We conclude that -adrenoceptors are situated close to the nerve endings and -adrenoceptors close to COMT sites. Since these results do not differ qualitatively in two vessels with different patterns of innervation, we conclude that this asymmetry in the distribution of -and -adrenoceptors may be due to either an uneven distribution of cells with only one type of receptors each or due to an uneven distribution of receptors on the same cell.     

Plasma concentration of α-methyldopa and sulphate conjugate after oral administration of methyldopa and intravenous administration of methyldopa and methyldopa hydrochloride ethyl ester

Summary The plasma concentrations of free -methyldopa and methyldopa sulphate conjugate were measured in 7 hypertensive patients with normal renal function following -methyldopa (1 g) orally. Five of these patients subsequently received -methyldopa ethyl ester (250 mg) (methyldopate) intravenously and two further patients received 250 mg of -methyldopa intravenously. After oral administration a large amount of total plasma -methyldopa was present as sulphate conjugate. There were wide interindividual differences in the ratio of free: conjugated -methyldopa in plasma (ratio at 4 hours ranged from 3.73 – 0.83) suggesting that individual differences in the extent of sulphate conjugation may occur. There was no close correlation between the degree of conjugation and the fall in arterial pressure. At all time intervals examined, plasma concentrations were higher following intravenous -methyldopa than -methyldopate. The plasma concentration of -methyldopa (free and esterified) 60 minutes after i.v. -methyldopate was 1.7±0.3 µg/ml wile at the same time after the same dose of methyldopa by the same route the mean concentration was 5.9 µg/ml. Although small amounts of sulphate conjugate were detected after i.v. -methyldopate, insignificant quantities of conjugate were found after i.v. -methyldopa. The average fall in mean arterial pressure was 27 mm Hg following i.v. -methyldopa but only 2.7 mm Hg following -methyldopate. These results suggest that sulphate conjugation of -methyldopa occurs in the gastrointestinal tract during absorption. Hydrolysis of -methyldopa ethyl ester does not appear to be instantaneous and pharmacokinetic differences between the ester and free -methyldopa have been demonstrated.     

Biodegradation of alpha-hexachlorocyclohexane

Summary Alpha-hexachlorocyclohexane (-HCH) is dechlorinated by enzymes contained in rat liver cytosol and microsomes. An evidence was obtained that in the cytosol there are two -HCH dechlorinating enzymes at least: one operates only in the presence of reduced glutathione (GSH) and catalyzes dechlorinations associated with the formation of another hydrophilic product. This product is probably a conjugate of the -HCH-residue with GSH. The other cytoplasmic -HCH-dechlorinase requires no additions. The microsomes, too, contain two -HCH dechlorinases at least: one is stimulated by GSH, the other by NADPH.Part V.: Naunyn-Schmiedeberg's Arch. Pharmacol. 288, 65–78 (1975)     

β2-Adrenergic Receptor Genotype-Related Changes in cAMP Levels in Peripheral Blood Mononuclear Cells After Multiple-Dose Oral Procaterol

Purpose. To evaluate the ₂-adrenergic receptor (₂AR) genotype frequency in the Japanese population and the relationship between ₂AR genotype at amino acid position 16 (₂AR-16) and desensitization to ₂-agonist ex vivo. Methods. The ₂AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the ₂AR-16 genotype and the desensitization to ₂-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50g, Meptin®) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). Results. Allele frequency was Arg16:Gly16 = 46%:54%, Gln27:Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. Conclusions. The desensitization to ₂-agonist was associated more frequently with the mutation at ₂AR-16 (Gly16).     

Age-related alterations in α1- and β-adrenoceptors mediated responsiveness of rat aorta

Summary We have examined the responsiveness of - and -adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA₂ value of 9.45, suggesting that the receptor is an ₁-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the -adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in ₁-adrenoceptor responsiveness in senescence, and a loss of -adrenoceptor-mediated responses in maturation. Send offprint requests to J. R. Docherty at the above address