Down-regulation of neurotensin receptors after ligand-induced internalization in rat primary cultured neurons.

When rat cultured neurons were incubated with unlabelled neurotensin (3 nM) for 1 or 24 h at 37 degrees C, the [3H]-neurotensin specific binding measured in cell homogenates was decreased to about 35 and 65% of control values, respectively. In these experiments, the decreases in binding corresponded to reductions of Bmax values without changes in the affinity. The slow neurotensin-induced receptor down-regulation is thought to result from receptor degradation since it was reduced by the lysosomotropic drugs chloroquine and methylamine and because no change in neurotensin mRNA level could be measured after the neurotensin stimulation. After their internalization, receptors slowly reappeared at the cell surface after further incubation in the absence of the peptide. Such receptor reappearance was prevented in the presence of the protein synthesis inhibitor cycloheximide and is therefore thought to result from new synthesis and not from recycling of internalized receptors. These results indicate that the neurotensin-induced receptor internalization in cultured neurons is irreversible and that it is followed by a down-regulation of the receptor through a degradative process.

Fractionation of human brain by differential and isopycnic equilibration techniques.

Fractionation of brain tissue by either differential or isopycnic centrifugation is a useful cytological and biochemical tool to study the intracellular localization of neuronal elements involved in neurotransmission. Several neuroreceptors and uptake sites were found to display a subcellular bimodal distribution in rat brain. However, in the human brain, little is known about the subcellular distribution of neurotransmitter receptors and amine uptake sites. Despite the inevitable post-mortem delay which seems to induce many more morphological changes than modifications of enzymatic or receptor distribution profile from the subcellular fractions, fractionation of human brain areas remains a valid procedure to explore the subcellular localization of neuronal constituents. This paper describes the methods used to separate human brain tissue. As we have previously demonstrated in rat and dog brains, our results indicate that differential and isopycnic fractionation techniques, used with a large number of markers such as enzymes, receptors and uptake sites, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

Subcellular distribution of receptor sites in human brain: differentiation between heavy and light structures of high and low density.

Studies of the subcellular localization of neuroreceptors in the rat brain have shown that most of them are associated with light and low density subcellular fractions. In two human brain areas, quite different subcellular distributions were observed. After fractionation by differential centrifugation of frontal cortex homogenates, benzodiazepine and serotonin 5-HT2 receptors were mainly found in the heavy mitochondrial (M) fraction, whereas mu-opiate and muscarinic cholinergic receptors were mainly concentrated in the microsomal (P) fraction. In human putamen, the presynaptic markers of dopaminergic nerve terminals (neurotensin receptors, dopamine uptake sites and amine vesicular transporter-binding sites), benzodiazepine receptors and serotonin uptake sites were recovered both in the high and low density fractions, whereas the muscarinic, opiate and, to a lesser extent, dopamine D2 receptors were mostly concentrated in the microsomal fraction. In the cerebral cortex, after isopycnic centrifugation in sucrose gradients, neuroreceptors were found in the high density fractions where the peaks of cytochrome oxidase and that of nerve endings, as identified by amine uptake and by means of electron microscopy were also found. A single peak of benzodiazepine receptors was observed in high density (1.15-1.17 g/ml) fractions suggesting that these receptors are much more concentrated in the nerve terminals or dendrites rather than in the dendritic spines or vesicles. The fact that muscarinic and opiate receptors were recovered in the P fraction with plasma membrane constituents and also in M and L fractions, which is confirmed by a bimodal distribution in sucrose gradient, suggests that they are localized in both the nerve terminals or dendrites and in the small vesicles or dendritic spines. In the putamen, much of the specific binding to uptake sites for dopamine and serotonin was recovered in the high density fractions, but the existence of another peak at a lower density indicates the presence of microsomal uptake sites. The results indicate that differential and isopycnic fractionation methods performed on human brain samples, make it possible to separate tissue fractions enriched in nerve endings, dendrites, dendritic spines, plasma membranes or vesicles.

Rapid agonist-induced decrease of neurotensin receptors from the cell surface in rat cultured neurons.

The regulation of neurotensin receptors was studied in vitro in primary cultures of neuronal cells. High affinity receptors for [3H]neurotensin were found in homogenates and at the cell surface of intact neurons cultured from the brain of rat embryos. When intact cells were incubated with 3 nM neurotensin (1-13), a rapid decrease in [3H]neurotensin binding was observed; about 60% of neurotensin receptors disappeared from the cell surface in less than 15 min. This corresponded to a reduction of the Bmax value without a change in the binding affinity. The decrease in neurotensin receptor number was also induced by the active fragment (8-13) of neurotensin but not by its inactive fragment (1-8). It was partially inhibited by bacitracin, at concentrations which are known to interact with receptor internalization, and was not detected when intact cells were incubated at 0-4 degrees with the unlabeled peptide. When intact neurons were incubated with [3H]neurotensin, there was a rapid ligand uptake and the kinetics of endocytosis were similar to those of the cell surface receptor disappearance. Once endocytosed, [3H]neurotensin could not be released (or displaced) from either intact neurons or homogenates, suggesting the sequestration of the labeled peptide in vesicles or other subcellular structures. Therefore, the present results suggest that the rapid agonist-induced decrease in the number of neurotensin receptors from the cell surface corresponds to an internalization process which involves a simultaneous receptor-mediated peptide endocytosis.

[3H]GBR 12935 binding to dopamine uptake sites: subcellular localization and reduction in Parkinson's disease and progressive supranuclear palsy.

[3H]GBR 12935 bound with high affinity to dopamine uptake sites in rat striatum where a close parallelism was observed between the subcellular localization profiles for [3H]dopamine uptake and [3H]GBR 12935 specific binding. Using the same ligand, we characterized the dopamine uptake sites in human striatum: the mean KD value was 3.2 nM and the specific binding was inhibited by several dopamine uptake blockers but with slightly lower affinities than those observed in the rat. The subcellular localization profile revealed a synaptosomal enrichment of the specific binding in human striatum. [3H]GBR 12935 binding was decreased in the putamen and caudate nucleus of subjects with Parkinson's disease (33 and 46% of control values, respectively) and progressive supranuclear palsy (38 and 57% of control values, respectively). It is very unlikely that the remaining binding sites in both diseases correspond to piperazine acceptor sites that are not involved in dopamine uptake. However, we cannot exclude the possibility that some of these remaining dopamine transporter sites are not functional, since the reduction in [3H]GBR 12935 specific binding was less marked than the decrease in the dopamine content of the same areas.

Regional and subcellular localization in human brain of [3H]paroxetine binding, a marker of serotonin uptake sites.

The characteristics of the binding of [3H]paroxetine, a selective serotonin (5-HT) uptake blocker, were investigated in human brain. The Kd value was 0.23 +/- 0.07 nM, and the Bmax value was 190 +/- 39 fmol/mg protein in the putamen. The capacity of various antidepressive drugs to inhibit [3H]paroxetine-specific binding in human brain was well correlated with their capacity to inhibit [3H]5-HT uptake in rat brain. The highest concentrations of [3H]paroxetine-specific binding sites were found in the substantia nigra, hypothalamus, and hippocampus. Lower values were obtained in the basal ganglia and the thalamus. The specific binding was very low in cerebral and cerebellar cortices. The regional distribution of [3H]paroxetine binding sites differs from that of [3H]ketanserin binding to S2 serotonin receptors. The subcellular distribution of the [3H]paroxetine-specific binding sites obtained by differential centrifugation revealed a synaptosomal enrichment in the frontal cortex and striatum, whereas an enrichment in the microsomal fraction was found in striatum. The results show that [3H]paroxetine is a ligand of choice to label the 5-HT uptake molecular complex in human brain.

Benzodiazepine receptors in normal human brain, in Parkinson's disease and in progressive supranuclear palsy.

Benzodiazepine receptors have been characterized in human brain. They have been localized mainly in the cerebral cortex and a synaptosomal enrichment was observed after brain fractionation by differential centrifugation. Benzodiazepine receptors were studied in Parkinson's disease and in progressive supranuclear palsy (PSP). In both diseases, the [3H]flunitrazepam specific binding was unchanged when compared to control groups (Bmax and KD values) except in the caudate nucleus of parkinsonian patients where an increase of the specific binding was observed. The subcellular distribution profile of benzodiazepine receptors in Parkinson's disease was similar to that of controls. gamma-Aminobutyric acid (GABA) still enhanced the [3H]flunitrazepam-specific binding (increase of binding affinity), indicating that the functional link between GABA and benzodiazepine receptors remained intact in Parkinson's disease. The present results suggest that benzodiazepine receptors in human striatum are localized on neuronal elements which do not degenerate in Parkinson's disease and PSP.

Benzodiazepine receptors in human brain: characterization, subcellular localization and solubilization.

1. Benzodiazepine receptors have been characterized in human brain mainly using [3H]-Ro 15-1788 and [3H]-flunitrazepam. Both ligands present a very high affinity for the receptor sites (Kd values of 0.56 and 1.53 nM respectively). 2. GABA enhanced the affinity of [3H]-flunitrazepam and [3H]-diazepam, but not that of [3H]-Ro 15-1788 and [3H]-methyl-beta-carboline 3-carboxylate for their specific binding sites as well in cerebral as in cerebellar human cortex. 3. Subcellular distribution of the benzodiazepine receptors revealed a main synaptosomal localization in human cerebral cortex, cerebellum and striatum. 4. Solubilized benzodiazepine receptors were obtained using 0.5% sodium deoxycholate and were characterized with [3H]-Ro 15-1788. The solubilized receptors are still coupled to GABA receptors since the [3H]-flunitrazepam specific binding was enhanced in the presence of micromolar concentrations of GABA.