Reproducibility of cerebral glucose utilization measured by PET and the [18F]-2-fluoro-2-deoxy-d-glucose method in resting,healthy human subjects

The stability of cerebral glucose utilization was examined in nine right-handed, healthy men (age, 24.88±2.93 years) using positron emission tomography (PET) and the [¹⁸F]-fluorodeoxyglucose (FDG) method. Each study was run twice at intervals of 1–12 weeks with the subject at rest. The average cerebral metabolic rate for glucose (CMRGlu) was 5.40±0.71 mg/100 g per min (coefficient of variance, 13.08). The average intraindividual variation of CMRGlu was 7.91%±15.46% (P=0.13). Metabolic indices (MI: regional/mean cortical CMRGlu) were used to determine the regional cerebral metabolic distribution. The interindividual (coefficient of variance, 7.13) and intraindividual variabilities (average variation, –0.12%±8.76%) of MI were smaller than those of metabolic rates. No reproducible significant asymmetry was observed. The FDG method used with subjects at rest thus yields low intraindividual variability of both cerebral glucose consumption and regional metabolic distribution, even at an interval of several weeks. Cerebral glucose utilization measured under such conditions may act as a reliable reference for determination of the influences of physiological (activation), pharmacological or pathological processes on cerebral glucose metabolism.Abbreviations FDG [¹⁸F]-fluorodeoxyglucose - CMRGlu cerebral metabolic rates for glucose - MI metabolic indices - EEG electroencephalogram - FWHM full width at half maximum     

Oxidative activation and inactivation of the carcinogen 2-acetylaminofluorene by various purified cytochromes P-450 from 3-methylcholanthrene pretreated rats

Ring- and N-hydroxylations of 2-acetylaminofluorene (AAF) have been examined in a reconstituted system with 4 purified hepatic microsomal cytochromes P-450 isolated from 3-methylcholanthrene (MC)-pretreated rats. Among these 4 isozymes, P-450D showed the most activity whereas P-450C was devoid of any activity; two other P-450s exhibited moderate activity. These and Ouchterlony's double diffusion analyses suggest involvement of multiple cytochromes P-450 in AAF oxidations.     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Solubilization of serotonin S2-receptors from human brain

Serotonin S₂-receptors were solubilized from human brain by means of the mild detergent, lysolecithin. Previous studies have shown that the serotonin S₂-receptors in human brain are mainly enriched in the cortex. A total particulate fraction from human cortex was treated with 0.25% lysolecithin. [³H]Ketanserin binding sites from the soluble extract showed the binding characteristics of S₂-receptors: potent 5HT antagonists like pirenperone, methysergide and pipamperone competed for [³H]ketanserin binding at nanomolar concentrations. The agonists bufotenin and serotonin themselves were more active than the potent dopamine agonist tetraline. Binding was saturable with a low K_D (1.07 nM) and reversible. There was a good correlation between the drug potencies in both soluble and membrane preparations and also with the IC₅₀ values previously obtained in membrane preparations and soluble extract from rat brain. Therefore, lysolecithin allows serotonin S₂-receptors from human brain to be obtained in a molecularly dispersed form with the same high affinity properties as in the original membranes.     

α2-Adrenoceptors in human forebrain: autoradiographic visualization and biochemical parameters using the agonist [H]UK-14304

The regional distribution and biochemical parameters ofα₂-adrenoceptors in post-mortem human brain tissue were analyzed in autoradiographic and membrane binding studies, using the full agonist, [³H]UK-14304. Autoradiographic visualization of these receptors in the forebrain revealed a heterogeneous anatomical distribution with high levels in the neocortex, ventral hypothalamus, hippocampus and some thalamic nuclei, among others. In membrane binding studies, analyses of saturation curves indicated the presence of a single population of sites for [³H]UK-14304 both in human and rat brain. There was a good correlation between the density of aα₂-adrenoceptors obtained by both procedures. [³H]UK-14304 is a feasible ligand to localise and quantifyα₂-adrenoceptors in human post-mortem material by autoradiographic and membrane binding techniques.     

Reversal by retinoids of keratinization induced by benzo[alpha]pyrene in normal hamster tracheal explants: comparison with the assay involving organ culture of tracheas from vitamin A-deficient hamsters

In a defined culture system for hamster tracheal explants, the activity of 12 different retinoids was evaluated for reversal of keratinization induced by exposure to the carcinogen, benzo[a]pyrene (BP-HTOC assay). The effects of retinoids in this system were compared to those in a defined culture system for tracheal explants from vitamin A-deficient hamsters (standard-HTOC assay). In both assays, all-trans-retinoic acid (RA) and 13-cis-RA were the most active retinoids. For RA and 13-cis-RA, the values of ED₅₀ determined in the BP-HTOC bioassay were 4 × 10⁻¹² and 1 × 10⁻¹¹ M, respectively, whereas the corresponding values in the standard HTOC assay were 2 × 10⁻¹¹ and 3.3 × 10⁻¹⁰ M. For all 12 retinoids, the ED₅₀ values from the BP-HTOC were lower than those from the standard-HTOC assay, and there was also a statistically significant correlation between the rank-ordering of ED₅₀ values from the 2 assays. Among 3 N-(retinoyl)amino acids examined in both assays, N-(retinoyl)leucine was the most active, N-(retinoyl)phenylalanine the least active, and N-(retinoyl)alanine intermediate. Among a novel series of bifunctional retinoic acid analogues, the dicarboxyl derivative was the most active. On the basis of these results, the BP-HTOC assay appears to be one of the most sensitive assays for retinoids yet developed. This assay is an appropriate model for evaluating the chemopreventive potential of new retinoids in vitro.     

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.     

Histamine H2 receptor overexpression induces U937 cell differentiation despite triggered mechanisms to attenuate cAMP signalling

Knowing that cell-surface receptors that recognize and respond to extracellular stimuli are key components for the regular communication between individual cells required for the survival of any living organism, the aim of the present work was to investigate the effect of H2R overexpression on the U937 signal transduction pathway and its consequences on cell proliferation and differentiation. The overexpression of H2R led to an increase in cAMP basal levels, a leftward shift of agonist concentration-response curves, and similar maximal response to agonist treatment, suggesting that overexpressed H2Rs act as functional spare receptors. In this system cells triggered several mechanisms tending to restore cAMP basal levels to those of the na?ve cells. H2R overexpression induced PDE activity stimulation and GRK2 overexpression. In spite of the onset of these regulatory mechanisms, H2 agonist and rolipram treatments induced the terminal differentiation of the H2R overexpressed clone, conversely to the na?ve cells. Present findings show that stably H2R overexpression alters cAMP signalling as the result of not only the amounts of second messenger generated but also the activation or upregulation of various components of signalling cascade, leading to an adapted biologically unique system. This adaptation may represent an advantage or a disadvantage, depending on the biological system, but in any case, the existence of compensatory mechanisms should be considered when a clinical treatment is designed.     

Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: a significant role of specific binding by serum albumin in the improved potency and stability

AS-3201 [(3R)-2'-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4'(1'H)-pyrrolo[1,2-a]pyrazine]-1',2,3',5(2'H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 (R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode (S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA-R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation. (13)C NMR observations of the succinimide ring (5-(13)C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and (19)F NMR of the pendent benzyl ring (2-(19)F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.