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J Bockaert

Publications and source records attributed to J Bockaert.

At least 271 records · Page 15Linked to original sources
PubMed1984

Topographical distributions of 32K and 48K cAMP-regulated phosphoproteins: relationships to dopamine and serotonin innervations in striatum, substantia nigra and cerebral cortex.

The topographical distribution of a cAMP-regulated phosphoprotein of an apparent molecular weight (Mr) of 32,000 (32K) was examined in homogenates prepared from microdiscs punched out from serial frozen slices of the striatum. The amount of this phosphoprotein progressively diminished from the rostral to the caudal part of the striatum as did both the dopamine-innervation and the dopamine (D1)-sensitive adenylate cyclase. After kainic acid lesion of the rostral part of the striatum, the 32K phosphoprotein disappeared in this area and we observed a 48% decrease in the amount of 32K phosphoprotein found in the substantia nigra. 6-Hydroxydopamine lesions of the nigro-striatal dopaminergic pathway did not affect the 32K phosphoprotein either in striatum or substantia nigra. These results suggest that in the nigro-striatal pathway, the 32K phosphoprotein is closely associated with dopaminoceptive neurons containing D1 receptors. In the cerebral cortex the association of 32K phosphoprotein with dopaminoceptive neurons is more questionable since we did not find a higher density of this phosphoprotein in areas containing a high amount of D1 receptor (frontal cerebral cortex) than in areas containing a low amount of D1 receptor (parietal cerebral cortex). In the course of this study we found another cAMP-regulated phosphoprotein of an Mr of 48,000 (48K). The amount of this phosphoprotein increased progressively from the rostral to the caudal part of the striatum, a pattern of distribution close to that of serotonin terminals. This protein was also present in the substantia nigra. Kainic acid lesioning of the rostral part of the striatum did not affect the amount of the 48K phosphoprotein within the substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The Ca2+/C1- dependent L-[3H]glutamate binding: a new receptor or a particular transport process?

Ca2+/C1- increases the L-[3H]glutamate binding to rat brain synaptic membranes. It was suggested that Ca2+/C1- expresses a new class of glutamate receptors. We report several lines of evidence suggesting that Ca2+/C1- in fact favours a glutamate transport into membrane vesicles. This finding may serve to reconcile most of the discrepancies found in the literature on the glutamate binding and its pharmacology.

Animals↗
PubMed1984

Solubilization of brain alpha-2 adrenoceptor with a zwitterionic detergent: preservation of agonist binding and its sensitivity to GTP.

Alpha-2 adrenergic receptors were solubilized from calf cerebral cortex using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The soluble extract retained the ability to bind the partial alpha 2 - adrenergic agonist [3H] p-aminoclonidine. This binding was of high affinity (KD = 1.5 +/- 0.3 nM) saturable (Bmax = 150 +/- 24 fmol/mg) and reversible. The orders of potencies of alpha-adrenergic drugs and nucleotides, for inhibition of binding, paralleled those previously observed on membrane bound alpha 2-adrenoceptors. Furthermore the very rapid decrease of [3H] p-amino-clonidine binding induced by GTP suggest that the guanyl nucleotide effect is due to an allosteric mechanism. These results indicate that in the soluble extract the interaction between the alpha 2-adrenoceptor and the GTP-binding regulator protein is preserved thus providing an interesting model to study the molecular structure of this system.

Animals↗
PubMed1984

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation.

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.

Cells, Cultured↗
PubMed1984

Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin-releasing factor.

Crude plasma membrane fractions were prepared from female Wistar rat anterior pituitaries. These fractions contained a single population of specific 3H-labeled [8-lysine]vasopressin [( 3H]vasopressin) binding sites with a dissociation of constant (Kd) of 8 +/- 2 X 10(-9) M and maximal binding capacity of 244 +/- 45 fmol/mg of protein. The Kd values for a series of vasopressin structural analogues with selective vasopressor or antidiuretic activities were determined together with the corresponding corticotropin-releasing activities (isolated perfused pituitary cells were used). A good correspondence was found between the two sets of values, suggesting that the detected vasopressin binding sites are the receptors involved in vasopressin-induced corticotropin release. The order of potency of these analogues for the binding to hypophysial receptors was similar to that found for the binding to the receptors involved in the vasopressor response. Corticotropin-releasing factor and angiotensin did not affect vasopressin binding to pituitary membranes. Median eminence extracts inhibited [3H]vasopressin binding with an efficiency very close to that expected from their vasopressin content. Corticotropin-releasing factor activated, and angiotensin inhibited, the adenylate cyclase activity of pituitary membranes. Under the same experimental conditions, vasopressin did not influence adenylate cyclase activity nor did it affect the corticotropin-releasing factor-induced activation. These data support the view that vasopressin is one component of the multifactorial regulation of corticotropin release and that it acts through a cAMP-independent pathway. The potentiation by vasopressin of corticotropin-releasing factor-induced cAMP accumulation in intact cells very likely proceeds through indirect mechanisms, which are not expressed in broken cell preparations.

Adenylyl Cyclases↗
PubMed1984

Turnover of adrenergic receptors under normal and desensitized conditions.

Alpha 1 and beta adrenergic receptor metabolism was investigated by studying receptor reappearance after an irreversible blockade. Phenoxybenzamine was used to irreversibly block alpha 1 adrenergic receptors both in vitro in the BC3H1 cell line and in vivo in rat submaxillary glands. In these two systems, the alpha 1 adrenergic receptor reappearance followed a monoexponential kinetic allowing to determine the half-life of the receptor (23h in vitro, 33h in vivo) as well as the rate of receptor synthesis and degradation. the receptor reappearance was due to receptor synthesis since it was blocked by cycloheximide. The irreversible blockade of beta adrenergic receptors was done with an alkylating beta adrenergic antagonist that we recently developed: Br-pindolol (1). This ligand has high efficiency and blocked at 10(-7)M 80-90% of the beta adrenergic receptors present in C6 glioma cells in culture. After this irreversible blockade, receptors reappeared only during cell division. At confluency, when cells did not significantly divide, receptor synthesis could hardly be detectable. Therefore, at confluency, the metabolic stability of the beta adrenergic receptor is considerable, compared to that of the alpha 1 adrenergic receptor. This stability was confirmed by the observation that after an almost complete "down-regulation" of the beta adrenergic receptor, receptor repopulation of the C6 glioma cells was total and occurred in the presence of cycloheximide.

Animals↗
PubMed1984

Hormonal inhibition of adenylate cyclase. A crucial role for Mg2+.

In several adenylate cyclase systems (anterior pituitary gland, human platelets, adipocytes, rat liver membranes), inhibitory hormones were shown to reduce basal adenylate cyclase activity by decreasing the "apparent affinity" of those systems for Mg2+ activation, without modifying the Vmax of the reaction. In the absence of hormones, the Mg2+ dose-activation curves were monophasic, whereas in the presence of hormones a clear heterogeneity was revealed. Therefore, inhibitory hormones induced a right-hand shift in the Mg2+ dose-activation curve. This hormonal effect was concentration-dependent. In human platelets, the inhibition of prostaglandin E1-stimulated adenylate cyclase by norepinephrine was also due to a decrease in the apparent affinity for Mg2+. In anterior pituitary gland, when Mg2+ was substituted by Mn2+, similar results were obtained. Thus, dopamine produced its inhibition by decreasing the apparent affinity for Mn2+ both under basal and vasoactive intestinal peptide-stimulated conditions. At Mg2+ or Mn2+ concentrations high enough to obtain saturation of the low apparent affinity state, hormone-induced inhibition was not observed. In anterior pituitary gland and in human platelet membranes, Na+ was not required in order to observe adenylate cyclase inhibition by catecholamines. In adipocytes and rat liver membranes, however, Na+ was required. In both systems, GTP was able to transform adenylate cyclase to a low Mg2+ apparent affinity state. Na+ was able to reverse (in a dose-dependent manner) the system to a high Mg2+ apparent affinity state. Once in this state, hormones were shown to inhibit adenylate cyclase activity by reverting the enzyme to a low apparent affinity state for Mg2+.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Adenylate cyclase inhibition by hormones. The Mg2+ hypothesis.

In washed anterior pituitary membranes, there is enough GTP to occupy Ns and therefore to obtain activation of adenylate cyclase by vasointestinal peptide. GTP concentrations needed to obtain adenylate cyclase inhibition by dopamine (above 5 X 10- M) stimulate the adenylate cyclase. The dopamine effect is a blockade of this stimulation. We propose that at least in this system, Ni does not inhibit but stimulates the adenylate cyclase and that inhibitory hormones block this stimulation. We also demonstrate in several adenylate cyclase systems that hormones produced adenylate cyclase inhibition by lowering their Mg affinity A general model for adenylate cyclase activation and inhibition is proposed.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Inhibition of brain adenylate cyclase by A1 adenosine receptors: pharmacological characteristics and locations.

When tested under conditions reducing the endogenous production of adenosine (presence of adenosine deaminase (ADA) 1.6 IU/ml; and deoxyadenosine triphosphate (d-ATP), and in the presence of both NaCl and GTP, the ADA-resistant analog phenylisopropyladenosine (PIA) inhibited the adenylate cyclase of several brain tissues. These tissues included: (1) 5 brain areas of adult rats (frontal and parietal cortex, cerebellum cortex, hippocampus and striatum)--hypothalamus and mid-brain adenylate cyclases were not inhibited by PIA; (2) astrocytes in primary cultures prepared from cerebral cortex of newborn mice; and (3) neurons in primary cultures prepared from striata of 15-day-old mouse embryos. The specificity profile of the adenosine receptor involved in the inhibition was determined in astrocytes. It was typical of an A1 adenosine receptor (high affinity of PIA; Ka app: 9 +/- 5 X 10(-9) M (n = 4) compared to the affinity of 5'-N-ethylcarboxamide adenosine (NECA); Ka app: 1.3 +/- 0.6 X 10(-7) M (n = 3). There was an excellent correlation between the affinities of several adenosine agonists and antagonists for A1 receptors coupled with an adenylate cyclase in astrocytes and for the receptors labeled with N6-cyclohexyl-[3H]adenosine in brain cortex. In adult rat striatum as well as in astrocytes and striatal neurons in culture the adenylate cyclase was inhibited by low PIA concentrations through A1 receptors and stimulated by higher concentrations through A2 receptors. In contrast, A2 receptors were not detected in adult rat cerebral cortex. In adult rat striatum, A1 and dopamine receptors coupled with an adenylate cyclase seemed to be located on different cell populations. In contrast, in astrocytes A1 and beta-adrenergic receptors coupled with adenylate cyclase were apparently located on the same cells.

Adenosine↗
PubMed1983

GTP-dependent anion-sensitive adenylate cyclase in snail ganglia potentiation of neurotransmitter effects.

Snail ganglia possess an anion-sensitive adenylate cyclase. This enzyme was stimulated 100% by chloride in a strictly GTP-dependent manner. The apparent affinity of chloride for adenylate cyclase was 2 X 10(-4) M. Halogens were found to be the most active anions. Some inorganic anions such as SO4(2-) and H2PO4- were inactive, as were all the organic anions tested. Stimulation was not cumulative for any maximal concentration of the active anions except fluoride. Chloride potentiated the effect of fluoride, indicating that the anion effect is not fluoride-like. Another striking result is that chloride enhanced adenylate cyclase sensitivity to the neurotransmitters serotonin and dopamine. The absence of chloride stimulation when Mg2+ was replaced by Mn2+ further indicates a role of the GTP-binding protein (the G/F unit). Chloride could reversibly stimulate the adenylate cyclase activity already maximally stimulated by guanyl 5'-imidodiphosphate. We therefore suggest that, in snail ganglia, chloride raises the activity of the G/F unit-catalytic unit complex at some stage after its formation. The same specific anion-sensitive adenylate cyclase was also found in some of the rat tissues tested.

Adenylyl Cyclases↗
PubMed1983

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.

Adenosine↗
PubMed1983

Differences between agonist and antagonist binding to alpha 1-adrenergic receptors of intact and broken-cell preparations.

Alpha 1-Adrenergic receptors of a nonfusing muscle cell line (BC3H1) have been identified on intact and broken-cell preparations by using the high-affinity antagonist [3H]prazosin. In intact cells, both equilibrium and kinetic studies gave KD values in the range of 0.07-0.12 nM. The maximal number of binding sites determined by Scatchard analysis was about 85,000 +/- 9,000 sites/cell. Antagonists bound to the [3H]prazosin binding sites with Michaelis-Menten characteristics, and their specificity was typical of alpha 1-adrenergic receptors. No significant modification of antagonist binding was observed either after cell disruption or by lowering incubation temperature to 4 degrees. In contrast, in intact cells at 37 degrees, agonist competition curves were shallow with Hill coefficients of less than 1. The heterogeneity of [3H]prazosin binding sites toward (--)-norepinephrine also appeared in [3H]prazosin saturation experiments carried out in the absence and in the presence of this agonist. After cell disruption, the EC50 values of agonist competition curves decreased and Hill coefficients were close to 1. When the temperature was lowered from 37 degrees to 4 degrees, the affinity for (--)-norepinephrine in intact cells increased dramatically by 10,000 times and the Hill coefficient of the competition curve was equal to unity. This affinity shift induced by temperature was not so important in broken-cell preparations (50 times).

Adrenergic alpha-Agonists↗
PubMed1983

Turnover in vivo of alpha 1-adrenergic receptors in rat submaxillary glands.

In submaxillary glands, vas deferens, and cerebral cortex, [3H]prazosin labeled one homogeneous population of alpha 1-adrenergic receptors having a KD of 0.1 nM. Intravenous injections of phenoxybenzamine blocked these receptors in a dose-dependent manner without changing the affinity of the remaining sites for [3H]prazosin. The phenoxybenzamine efficiency was highest in submaxillary glands: 1 mg/kg completely blocked the alpha 1-adrenergic receptors but did not affect alpha 2-adrenergic, beta-adrenergic, and muscarinic receptors in this organ. After this blockade, the alpha-adrenergic receptors reappeared in the glands following a monoexponential time course. Analysis of this time course allows the determination of the rate constant for receptor degradation (k = 0.02 hr-1) and the rate of receptor production (r = 1.86 fmoles/mg of protein per hour). The half-life of the receptor was 33 hr. The reappearing receptors corresponded to newly synthetized receptors since their reappearance was blocked by i.p. injections of cycloheximide. Blockade of alpha 1-adrenergic receptors with phenoxybenzamine (2 mg/kg) did not affect receptor reappearance. In contrast, higher doses (4-20 mg/kg) decreased the velocity of receptor reappearance.

Animals↗
PubMed1983

Pharmacological characterization of the D2 dopamine receptor negatively coupled with adenylate cyclase in rat anterior pituitary.

In male and female rat anterior pituitary homogenates dopamine inhibited basal adenylate cyclase by 30% and 50%, respectively. Dopamine also inhibited vasoactive intestinal peptide-stimulated adenylate cyclase by 50% in both sexes. Sulpiride, a specific D2 antagonist, stereospecifically blocked with high affinity the dopamine inhibition in both males and females. RU 24926, a specific, non-catechol, non-ergot D2 agonist, also inhibited basal adenylate cyclase of female pituitary with a higher apparent affinity than dopamine (KDapp 20 nM and 450 nM, respectively). This effect was also stereospecifically antagonized by sulpiride. Apomorphine was also more potent (KDapp 100 nM) than dopamine, whereas norepinephrine and SKF 38393, a specific D1 agonist, were poorly active; isoproterenol and clonidine were inactive. Ergots derivatives such as CB 154, LY 14865, pergolide, and lergotrile were potent agonists. alpha-Dihydroergocryptine was a partial agonist of the dopamine receptor negatively coupled with an adenylate cyclase. Because of the slow association kinetics of this drug with the dopamine receptor, its KDapp (0.7 nM) for adenylate cyclase inhibition could be correctly determined only after a 30-min incubation period. All classical dopaminergic antagonists blocked dopamine inhibition of pituitary adenylate cyclase, pimozide (KI 1 nM) and spiperone (KI 0.8 nM) being the more potent. There were good correlations between the affinities of large series of agonists and antagonists for the anterior pituitary dopamine receptors negatively coupled with an adenylate cyclase on one hand, and for either D2 dopamine receptors labeled with [3H] dihydroergocryptine or [3H]spiroperidol in both pituitary and striatum, or D2 pituitary receptors involved in prolactin secretion on the other hand. It is concluded that the pituitary dopamine receptors negatively coupled with an adenylate cyclase are the classical D2 receptors involved in prolactin secretion.

Adenylyl Cyclases↗
PubMed1982

Characteristics and metabolism of alpha 1 adrenergic receptors in a nonfusing muscle cell line.

The BC3H1 nonfusing muscle cell line possesses binding sites for [3H]prazosin. These binding sites are typically alpha 1 adrenergic receptors as shown by their greater affinity (3700-fold) for prazosin than for yohimbine. Both kinetic and equilibrium analyses indicated that [3H]prazosin interacted with only one category of independent binding sites with the following characteristics. KD = 0.13 +/- 0.01 nM. Bmax = 97 +/- 5 fmol/mg of protein corresponding to 25,000 sites/cell (n = 17). Biosynthesis of the alpha 1 adrenergic receptor was investigated at cell confluency (when the number of cells and their total protein content were constant). Phenoxybenzamine (10(-9) M) irreversibly blocked 50% of the alpha 1 receptors in intact cells. More than 95% blockade of receptors was obtained with 10(-7) M phenoxybenzamine. After this blockade, new alpha 1 adrenergic receptors reappeared in the cells with monoexponential kinetics. These new receptors corresponded to synthesized receptors since their appearance was blocked by cycloheximide (1 micrograms/ml). The cycloheximide action was reversible. If one makes the simple and probable hypotheses that the receptor production is constant and that degradation is a monoexponential process, the analysis of the kinetics of reappearance allows the determination of the rate constant for receptor degradation (k = 0.03 h-1) and the rate of receptor production (r = 3.2 fmol/mg/h) corresponding to the synthesis of about 760 receptors/cell/h. The half-life of the receptor was 23 h.

Animals↗