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Notes: Abstract: The compound [3H-Tyr1,D-Ala2,Lcu-OH5]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase (enkephalinase) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [3H]Tyr-D-Ala-Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly3-Phe4 amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5-enkephalin levels.

Notes: a-Fluoromethylhistidine (α-FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)-α-FMH, brain HD activity was inhibited in a time-dependent and concentration-dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α-FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)-α-FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)-α-FMH did not alter histamine-N-methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α-FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.

Notes: Abstract: Marked reductions in opiate receptor binding (−42%), “enkephalinase” activity (−39%), and Met5-enkephalin levels (−72%) accompanied the well-established dopamine depletion in the substantia nigra pars compacta of Parkinsonian subjects. In contrast, enkephalinergic markers were not significantly modified in caudate nucleus.

Notes: The properties of the histamine-forming enzyme in human brain samples were studied utilizing a radiochromatographic procedure. The influence of postmortem conditions was checked with rat brains, and the results indicated that the enzyme activity is not altered in situ for a delay not exceeding 4 h at ambient temperature. Moreover, tissue blocks or homogenates can be stored at low temperatures for up to 3 months with a good preservation of the enzyme activity. The data indicate that histamine synthesis in the human brain involves the „specific” histidine decarboxylase (HD, EC 4.1.1.22) and not the aromatic l-amino acid decarboxylase; (1) the optimum pH is 7.4 at 10-6m-l-histidine; (2) the apparent Km is about 3.10-5m; (3) it is inhibited by α-hydrazino histidine and brocresine but not affected by α-methyl DOPA. Moreover, a major portion of the enzyme is localized in a subcellular fraction containing nerve terminals and it shows an uneven regional distribution which parallels that observed in the brain of other mammalian species. Taken together these data strongly suggest that histamine could play a neurotransmitter role in the human brain.

Notes: [Auszug] The addition of serotonin to slices from mouse cerebral cortex preincubated with 3H-glucose for 30 min, at a time when 3H-glycogen level in tissue has reached a plateau19, results in rapid hydrolysis of the 3H-polymer, The serotonin-induced gly-cogenolysis is concentration dependent, occurs with a ...

Notes: [Auszug] Thiorphan was designed to interact with critical residues of the active site of enkephalinase, as postulated on a hypothetical model (Fig. 1). The model was developed on the assumption that binding of enkephalins to this active site occurs in a manner analogous to that established at the molecular ...

Notes: [Auszug] The involvement of both HI- and H2-receptors in the cyclic AMP response to histamine in hippocampal slices was shown mainly by the biphasic rightward shift of concentration-response curves to this amine in the presence of low concentrations of mepyramine, a pure Hi-antihistamine and the additive ...

Notes: Summary Dopamine receptors subtypes were studied in homogenates from rat brain areas, mainly the corpus striatum, using the two highly selective ligands 3H-apomorphine and 3H-domperidone. The clearly biphasic inhibition of the specific binding of these two ligands by some agents allowed us to define four distinct classes of binding site. 3H-apomorphine labels two classes of site displaying a large difference in affinity for domperidone, i.e. class I sites well recognized (IC50=5 nM) and class II sites poorly recognized (IC50=10 μM). 3H-domperidone also labels two distinet classes of site displaying a large difference in affinity for apomorphine and dopamine, i.e. class III sites well recognized by these agents (IC50=5 and 35 nM, respectively) and class IV sites poorly recognized (IC50=790 nM and 14 μM, respectively). The two classes I and III represent a single pharmacological class of dopaminergic receptors (labelled by either 3H-apomorphine or 3H-domperidone) as indicated by 1) their almost identical pharmacological specificities (high correlation between K d or K i values for a variety of dopaminergic agonists and antagonists); 2) their similar capacity in striatum as well as in other brain regions; 3) the identical decrease in capacity following kainate lesions; 4) their similar sensitivity to GTP and thermal denaturation. Because the pharmacological specificity of these sites excludes the possibility that they represent the recognition sites of the dopamine-sensitive adenylate cyclase, i.e. D-1 receptors, we propose to term them D-2 receptors. Class II and IV sites also differ from D-1 receptors as shown by drug specificity and the effect of kainate. We propose to term class II sites D-3 receptors and class IV sites D-4 receptors. D-2 receptors are characterised by a high affinity for both dopamine receptor agonists and antagonists (K i and K d values in the nM range). They are localised post-synaptically to dopaminergic terminals in the striatum as indicated by 1) their decreased number (−60%) following kainate lesions of intrinsic neurones, and 2) their increased number (+40%) after 6-OHDA-induced degeneration of dopaminergic neurones. The capacity of D-2 receptors is decreased by 80% in the presence of 25 μM GTP. The binding of ligands to D-2 receptors preicubated at 45°C decreases with a half-life of 10 min. D-2 receptors may mediate behavioral actions of apomorphine in low dosage which are easily antagonised by neuroleptics. D-3 receptors appear to be, at least in part, autoreceptors: their number decreases in striatum after 6-OHDA lesions (−30%) and is not modified following kainate lesions. They are characterised by a high affinity (K i in the nM range) for dopaminergic agonists (except for bromocriptine) contrasting with a rather low affinity for antagonists. The pharmacologically homogeneous class of D-3 receptor appears heterogeneous regarding both localisation and regulation by GTP. D-4 receptors are partly localised on intrastriatal neurones (−17% after kainate lesions, +17% following 6-OHDA lesions). However, the small change after kainate-induced lesions suggests that a significant fraction of D-4 receptors is localised on terminals from extrinsic neurones. D-4 receptors are characterised by a high affinity for dopamine receptor antagonists (K i in the nM range) contrasting with a relatively low affinity for agonists. The number of D-4 receptors increases after either GTP or heat denaturation, a change which probably corresponds to the decrease in D-2 receptors. D-4 receptors may mediate typical behavioral actions of apomorphine in moderate dosage.

Notes: Summary The experimental conditions allowing to elicit by administration of dopamine agonists a climbing behavior in rats, apparently analogous to the stereotyped cage climbing behavior previously described in mice (Protais et al. 1976), have been established. Among the various strains of rats studied i.e. Sprague-Dawley, Long Evans and Wistar, the latters were selected as the most responsive to the dopamine agonist apomorphine. However, even in the Wistar strain, only about 60% of animals responded to a test-dose of 0.4 mg/kg apomorphine by adopting in a sustained manner the typical upright position against the walls of a suitable experimental cage. Hence responsive rats were preselected 4 days before the experimental sessions and finally rated during a 60-min observation period. Increasing the test-dose of apomorphine led to a biphasic effect, the spontaneous climbing behavior being decreased at low dosage and, then, both the percentage of climbing animals and the duration of the behavior were progressively increased at higher dosages. A scoring system based on an all-or-none evaluation of the frequency of stereotyped climbing episods over the 1 h observation period was finally adopted allowing to establish dose response curves to apomorphine and its more potent derivative N-propylnorapomorphine. Dexamphetamine (associated to L-Dopa) also produced the stereotyped climbing behavior. The latter was completely abolished in animals treated with the “atypical” antipsychotic sulpiride. The effects of lesioning various cerebral dopaminergic areas on the apomorphine-induced behavior were investigated. The response was not significantly altered following bilateral thermocoagulations of the striatum (restricted lesions), globus pallidus, nucleus interstitialis of the striae terminalis, amygdala, nucleus lateralis septi or nucleus accumbens. Intrastriatal injections of 6-hydroxydopamine were without significant effect while large striatal thermocoagulations abolished the stereotyped behavior but were accompanied by a profound motor deficiency. Bilateral lesioning of the substantia nigra by either thermocoagulation or 6-hydroxydopamine, the latter leading to a 60% decrease in 3H-dopamine uptake in striatum without significant modification in nucleus accumbens, resulted in a hypersensitive response to apomorphine. Hence, these experiments do not allow to identify a single brain area where dopamine receptors mediating the climbing behavior are located although they suggest that the receptors involved receive a dopaminergic input originating from the substantia nigra.

Notes: Summary Dopaminergic binding sites were studied in slices from rat striatum incubated in a physiological medium and using the two highly selective ligands 3H-apomorphine and 3H-domperidone. The clearly biphasic or stretched inhibition of the specific binding of these two ligands by domperidone or apomorphine, respectively allowed to define three distinct classes of binding site. It was demonstrated, by comparing the binding of the 3H-ligand added at the beginning of slice incubation or just before homogenisation of tissue and filtration, that the “specific” bindings only occurred during the incubation of slices. The inhibition constants (K i values) of dopaminergic agents for the three classes of binding site as also the dissociation constants (K d values) of 3H-ligands and the maximal capacity (B max) of the three classes of binding site were closely similar to those of binding sites previously demonstrated on rat striatal membranes, namely D-2, D-3 and D-4 sites (Sokoloff et al. 1980a, b). Their identification on a preparation in which the cellular organisation is largely preserved rules out the possibility that these sites represent an artifact due to membrane preparation. Unexpectedly the addition of guanyl nucleotides like GTP or GppNHp to the slice preparation decreased the binding of 3H-apomorphine to the high affinity sites (particularly to the D-2 sites) while D-4 site binding was correspondingly increased. The guanylnucleotide effect apparently took place before cell disruption and occurred at concentrations similar to those required in striatal membrane preparations. These observations, together with those indicating the presence of high affinity binding sites for dopaminergic agonists in intact striatal cells, suggest that a putative nucleotide regulatory unit of dopamine receptors, is not fully occupied by intracellular GTP but could be interacted with from the external face of the cell membrane.