J. Bockaert

Topographical distribution of dopaminergic innervation and of dopaminergic receptors in the rat striatum. II. Distribution and characteristics of dopamine adenylate cyclase--interaction of d-LSD with dopaminergic receptors.

The characteristics of dopamine adenylate cyclase in the rat striatum were first studied on homogenates of fresh tissues. In the assay conditions used, dopamine (10(-4) M) stimulated the enzyme activity by 250%. This effect was completely blocked by fluphenazine (10(-5) M; Ki=9X10(-9) M) and by phentolamine (10(-5) M; Ki=3 X 10(-7) M). D-LSD stimulated the adenylate cyclase activity (Km=1.4 X 10(-7) M) by interacting with dopamine receptors; indeed the dopamine effect on the enzyme activity was competitively reduced in presence of D-LSD. L-Isoproterenol (Km=10(-6) M) activated an adenylate cyclase through a receptor distinct form the dopaminergic receptor; this stimulation was not affected by fluphenazine or phentolamine but suppressed by DL-propranolol (10(-4) M). The topographical distribution of the dopamine, D-LSD and L-isoproterenol adenylate cyclase activities were examined in homogenates prepared from discs punched out on serial frozed (--7C) slices of the striatum. Under this condition, tge dioanube naxunak stunykatuib was if 150%. A 4.8-fold progressive decrease in the amount of cyclic AMP produced in presence of dopamine (10(-4) M) was observed in the rostrocaudal plane of the structure; the decline of the basal activity was 3.6-fold. The topographical curves of maximal activation of adenylate cyclase by dopamine and D-LSD were superimposable confirming that D-LSD acts on dopaminergic receptors. This topographical distribution of dopamine sensitive adenylate cyclase is comparable on one hand to that of endogenous dopamine and on the other hand to that of the dopamine high affinity uptake activity measured in simultaneous experiments. In contrast to that observed with dopamine or D-LSD, the topographical distribution of the adenylate cyclase sensitive to L-isoproterenol was homogenous within the striatum.

The effects of quipazine on 5-HT metabolism in the rat brain

SummarySince quipazine is a potent 5-HT agonist in peripheral organs, its possible stimulatory effects on serotoninergic receptors in the rat brain were investigated. Quipazine administration (10 mg/kg, i.p.) induced a significant decrease in the synthesis and turnover rates of serotonin in the brain stem as well as in the forebrain. It is not likely that these changes were mediated by a negative feed-back mechanism triggered by adirect action of quipazine on central 5-HT postsynaptic receptors. Indeed, in contrast to LSD and 5-methoxy-N,N-dimethyltryptamine, this compound failed to activate the 5-HT sensitive adenylate cyclase in colliculi homogenates of newborn rats. However, quipazine exerted direct effects on serotoninergic terminals. It inhibited competitively the reuptake process in synaptosomes (Ki =1.38×10−7 M) and stimulated the K+ evoked release of newly synthesized3H-5-HT in slices of the brain stem. Injected in vivo in a dose which affected 5-HT uptake and release, quipazine did not modify MAO activity. However, this activity was noncompetitively inhibited by high concentration of the drug in vitro (Ki=3.0×10−5 M). These actions are very likelyindirectly responsible for the stimulation of central 5-HT receptors.

Serotonin-sensitive adenylate cyclase and [3]serotonin binding sites in the CNS of the rat—I: Kinetic parameters and pharmacological properties

The 5-HT receptor linked to adenylate cyclase and the high affinity binding site for [3H]-5-HT were compared on the basis of their kinetic and pharmacological properties in the CNS of new born rats. Under normal assay conditions, the apparent affinity of 5-HT for its specific binding sites (Kd = 1−2 nM) was much higher than that for the receptor coupled to adenylate cyclase (KA app = 0.5−1.0 μM). When measured under the conditions of the cyclase assay, the apparent Kd for the binding was increased to 11.9 nM, a value which is still more than 40 times lower than the KA app characterizing the activation of adenylate cyclase by 5-HT. GTP affected both the binding of [3H]-5-HT and the 5-HT-sensitive adenylate cyclase. Guanyl nucleotides appeared to be essential for the activation of adenylate cyclase by 5-HT as 5-HT was inactive in a preparation of washed membranes unless added in the presence of GTP or GppNHp. In whole homogenates, GTP increased the affinity of 5-HT for the receptor-adenylate cyclase complex (KA app = 0.33μM in the presence of 10μM GTP). The specific binding of [3H]-5-HT was reduced by GTP and GppNHp but not GMP or ATP. However, the range of concentrations inducing a significant effect (⩾0.10mM GTP) was far higher than those which increased the 5-HT-induced activation of adenylate cyclase. There was little in common between the pharmacological profiles of the two systems. A group of 5-HT agonists containing a piperazine heterocycle [1- (m-trifluoromethylphenyl) piperazine, quipazine and MK-212] effectively displaced [3H]-5-HT from its binding sites but exerted no action on the 5-HT-sensitive cyclase, affecting neither the basal nor the 5-HT-stimulated cAMP production. Likewise, there was no correlation between the respective potencies of a series of 5-HT antagonists for inhibiting the binding of [3H]-5-HT and the 5-HT-induced cAMP production. These data suggest that the 5-HT receptor linked to adenylate cyclase is not identical with that which is measured by the binding of [3H]-5-HT and, thus, provide evidence for the possible existence of multiple receptors for 5-HT in the rat brain.

Is the dopamine sensitive adenylate cyclase in the rat substantia nigra coupled with 'autoreceptors'?

The neurones, containing dopamine, which originate in the substantia nigra (SN) and project in the striatum constitute the nigro-striatal dopaminergic pathway. The dopamine (DA) released from the dopaminergic nerve terminals interacts with a postsynaptic dopaminergic receptor which is coupled with a specific dopamine sensitive adenylate cyclase [l-6] . This adenylate cyclase has a topographical distribution similar to that of dopaminergic terminals [6,7] and remains after degeneration of the dopaminergic nigro-striatal pathway. From pharmacological data, it has been proposed that dopaminergic terminals in the striatum possessed another categoric of dopaminergic receptors also called ‘autoreceptors’ [8] since they are sensitive to the neurotransmitter of the neurone on which they are localized. They are involved in the control of dopamine synthesis [8] and release [9,10]. These autoreceptors are widely distributed in all the different parts of the dopaminergic neurones and especially on the cell bodies in the substantia nigra [ 1 l-l 41. The present study was done in order to know if the autoreceptors of the substantia nigra are coupled with an adenylate cyclase in the same way as the striatal postsynaptic dopaminergic receptors. For this purpose, the substantia nigra was dissected on frozen slices (-7°C) and homogenates prepared: they were found to contain a dopamine sensitive adenylate cyclase. This adenylate cyclase was also stimulated by Lnorepinephrine and to a lesser extent by apomorphine. Various neuroleptics which blocked the striatal dopamine sensitive adenylate cyclase were also able to inhibit competitively the nigral dopamine sensitive adenylate cyclase. The dopamine sensitive adenylate

Is dopamine-sensitive adenylate cyclase involved in regulating the activity of striatal cholinergic neurons?

SummaryThe dopamine (DA)-receptor mediated changes in striatal acetylcholine (ACh) levels have been studied to determine if this effect involves a D1-(adenylate cyclase dependent) or D2-(not linked to an adenylate cyclase) type of DA-receptor. Various DA-agonists (apomorphine, N-diphenethylamine derivatives) increased striatal ACh levels in both intact and 6-OHDA lesioned rats whereas only apomorphine stimulated the adenylate cyclase activity of striatal homogenates. The N-diphenethylamine compounds (RU 24213, RU 24926 and RU 26933) were without effect either on basal or DA-stimulated activities of this enzyme. In contrast, D-LSD (which acts as a partial agonist of the striatal DA-sensitive adenylate cyclase) did not modify the striatal ACh content. More interestingly, an intrastriatal injection of cholera toxin greatly stimulated striatal adenylate cyclase without altering ACh concentrations. Both haloperidol and methergoline antagonized the DA stimulation of adenylate cyclase, but only haloperidol decreased striatal ACh levels. These results indicate that the DA receptor involved in regulating the activity of striatal cholinergic neurons is of the D2-type.

Characteristics of the β1- and β2-adrenergic-sensitive adenylate cyclases in glial cell primary cultures and their comparison with β2-adrenergic-sensitive adenylate cyclase of meningeal cells

The agonist specificity pattern of the β-adrenergic adenylate cyclase in glial primary cultures was not typical of either β1- or β2-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both β1- and β2-adrenergic-sensitive adenylate cyclase (58 ± 17% and 42 ± 17% respectively). Similar properties of β1- and β2-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective β1 antagonist) (55 ± 10% and 45 ± 10% of β1- and β2-sensitive adenylate cyclase respectively). The curves for displacement of [3H]dihydroalprenolol by practolol confirm these results. For purpose of comparison, the β-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained β2-adrenergic receptors.

Tritium labelling of 8-lysine vasopressin and its purification by affinity chromatography on sepharose bound neurophysins.

The antidiuretic hormone vasopressin is a nonapeptide containing one disulfide bridge and tyrosyl and phenylalanyl residues in position -2 and -3, respectively. 8-Lysine vasopressin (LVP) is found in pig and hippopotamus while 8-arginine vasopressin (AVP) occurs in several other mammals (man, ox, rat...). These pituitary polypeptides exert selective effects on the permeability of the distal part of the mammalian nephron and on the active sodium transport and passive permeability to water and other low molecular weight molecules of the amphibian skin and bladder epithelial cells. These effects have been shown to result from a specific activation of an adenyl cyclase located within the membrane of the target cells (for review, see for instance [ 1 ] ). Either LVP or AVP is associated with oxytocin, a related peptide, and carried in the post-hypophysis by a family of relatively small proteins, the neurophysins [2], with which they form non covalent complexes [3]. The isotopic labelling of polypeptidi¢ hormones has been successfully applied, in this laboratory, to oxytocin [4], angiotensine II [5] and other compounds [6]. The principle is an iodination of the tyrosyl (or histidyl) residue(s) followed by catalytic substitution of the peptide bound iodine with tritium. In this report we describe a similar procedure which produces, after purification by affinity chromatography on a column of bovine neurophysins immobilized on Agarose, a highly radioactive compound (10 Ci/mmole) which exhibits all the biological and biochemical properties of the native hormone. This, obviously, provides a very useful tool for the examination of the molecular mechanisms by which this important biological compound interacts with neurophysins [7] and biological membranes.

Neuronal, glial and meningeal localizations of neurotransmitter-sensitive adenylate cyclases in cerebral cortex of mice

The neurotransmitter-sensitive adenylate cyclases, respectively present in the dissociated cells of new-born mouse cerebral cortex (containing both neuronal and glial cells) and in a homogeneous population of glial cells, were compared. The dissociated cells from the cerebral cortex of new-born mice were found to contain Ca2+-, dopamine-, serotonin- and purinergic-sensitive adenylate cyclases. The dopaminergic receptor involved was extensively characterized and was similar to that described in adult animals. Beta-adrenergic-sensitive adenylate cyclase was present but was poorly active. After 3 weeks in culture, the neurons disappeared and a homogeneous population of glial cells was obtained (96% of the cells synthetized glial fibrillary acidic protein). These glial cells contained a highly potent beta-adrenergic-sensitive adenylate cyclase, and adenosine- adn Ca2+-sensitive enzymes. Ca2+ stimulation of the adenylate cyclase was due to the presence of calmodulin. We suggested that the dopaminergic- and serotoninergic-sensitive adenylate cyclases which disappeared during culture are probably localized in neuronal cells. The presence of Ca2+-, adenosine- and beta-adrenergic-sensitive adenylate cyclases in glial cells does not exclude their presence in neuronal cells. For comparison, the same experiments were conducted on meningeal layers of new-born mice and on meningeal cells in culture. They both contained beta-adrenergic- and purinergic-sensitive adenylate cyclases.

Biogenic amines and adenosine-sensitive adenylate cyclases in primary cultures of striatal neurons

Primary cultures of virtually pure striatal neurons from 16-day-old mouse embryos can be obtained using a serum-free chemically defined medium. Membranes prepared from these cells contain dopamine, beta-adrenergic, serotonin and adenosine sensitive adenylate cyclases. The pharmacological properties of the dopamine receptors are similar to those found for D1 receptors in adults except for the apparent affinities for agonists which were 5-10 times higher in fetal neurons. Beta-adrenergic receptors of striatal and cerebellar fetal neurons are of the beta 1-subtype as indicated by their identical affinity for adrenaline and noradrenaline and by their homogeneous, high affinity for practolol (Ki = 1.3 X 10(-6)M). Adenosine and serotonin sensitive adenylate cyclases present classical characteristics. An extensive study of the additive effects of the 4 neurotransmitter-sensitive adenylate cyclases indicates that: (1) part of the neurons bear more than one type of biogenic amine receptors; (2) the serotonin receptors are always associated with adenosine receptors on the same neurons; (3) adenosine- and dopamine-sensitive adenylate cyclases are additive. From this it can be concluded that as far as their adenylate cyclases-linked amine receptors are concerned, a maximal number of 15 types of neurons are present in these striatal cell cultures.

Paradoxical decrease of brain 5-HT turnover by metergoline, a central 5-HT receptor blocker

SummarySince metergoline (1-methyl-8-beta-carbobenzyloxy-aminomethyl-10-alpha-ergoline) is a potent 5-HT antagonist in peripheral organs, its possible blocking effects on 5-HT receptors in the rat brain were investigated. In vitro, metergoline inhibited both the specific high affinity binding of 3H-5-HT onto synaptosomal membranes (IC 50=18 nM) and the stimulating effect of 10 μM 5-HT on the adenylate cyclase activity in colliculi homogenates from newborn rats (IC 50=12 μM). In vivo, the administration of metergoline (10 mg/kg i.p., 60 min before death) resulted in a significant decrease in the 3H-5-HT binding capacity of synaptosomal membranes from the forebrain of adult rats. Taken together, these data clearly indicated that metergoline is a potent blocker of some serotoninergic receptors in the rat brain. Surprisingly, the changes in 5-HT turnover occurring in the brainstem and in the forebrain 1 h after metergoline (2–10 mg/kg) treatment were similar to those normally induced by a central 5-HT agonist: both the rate of 5-HT utilisation and that of 5-HT synthesis were significantly decreased. These changes were in contrast to the acceleration of 5-HT turnover induced by the administration of another potent central 5-HT antagonist, methiothepin. These results are discussed in relation to the possible existence of several types of serotoninergic receptors in the rat brain. It is possible that the positive feedback regulation of 5-HT turnover is triggered by the blockade of serotoninergic receptors sensitive to methiothepin, but not to metergoline.