Werner De Potter

[3H]Rauwolscine behaves as an agonist for the 5-HTa receptors in human frontal cortex membranes

The alpha 2 adrenergic antagonist [3H]rauwolscine binds with comparable nanomolar affinity to alpha 2 adrenoceptors and the nonadrenergic 5-HT1A receptors sites in human frontal cortex membranes. Addition of 0.5 mM GTP into the incubation medium produces a significant decrease in the amount of [3H]rauwolscine binding sites (Bmax = 230 +/- 16 and 115 +/- 11 fmol/mg protein in the absence and presence of GTP, respectively). The affinity for [3H]rauwolscine remains unchanged (i.e. KD = 40 +/- 0.9 nM and 4.1 +/- 1 nM). This effect of GTP can be attributed to decreased binding of the radioligand to the 5-HT1A receptors. GTP decreases binding of [3H]rauwolscine to nearly the same level as the one corresponding to the alpha 2 adrenoceptors in membranes from both the human frontal cortex and hippocampus. The venom of the marine cone snail, Conus tessulatus, preferentially inhibits [3H]rauwolscine binding to 5-HT1A receptors as compared with the alpha 2 adrenoceptors. Following complete masking of the 5-HT1A receptors by this venom. GTP no longer affects the saturation binding characteristics of [3H]rauwolscine for the remaining alpha 2 adrenoceptors. Nucleotides decrease the binding of [3H]rauwolscine to the 5-HT1A receptors with an order of potencies (i.e. GTP gamma S greater than GPP(NH)P much greater than GDP greater than GTP much greater than ATP) that is typical for nucleotide-mediated receptor-G protein dissociation. This suggests that [3H]rauwolscine is a 5-HT1A receptor agonist and this conclusion is compatible with earlier functional studies, indicating that rauwolscine (as well as yohimbine) has agonistic properties at the level of 5-HT autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent in vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis

The adrenal medullary cell secretes catecholamines by exocytosis [3,8], a process in which secretory vesicles release their contents into the extracellular space upon fusion with the cell membrane. A similar system has been discovered for several endocrine, exocrine and neuronal cell types [3,11 ,17,20,22]. An in vitro interaction between secretory vesicles and their plasma membrane, followed by release of their soluble vesicular contents, has already been described for neurohypophyseal vesicles [ 121 and insulin granules [6]. These release systems show a definite Ca’+ dependence. Ca’+ also plays a critical part in the adrenal medullary stimulus-secretion coupling; cell membrane depolarisation causes an influx of Ca2+ which initiate the exocytosis of chromaffin granules [9]. Developing a cell-free model would be useful to investigate the events which occur between Ca2+ influx and exocytosis. [6] and [ 151 report, respectively, catecholamine release and fusion when adrenal medullary cell membranes are incubated with chromaffm granules. Here, we report that this interaction results in the release of other soluble granular components such as dopamine-P-hydroxylase (DBH, EC 1.14.17.1) and ATP together with catecholamines. This release is caused by PM Ca2+ levels and can be blocked by the addition of EGTA to the incubation medium. These data suggest that the in vitro interaction between chromaffin granules and their plasma membranes could be a simple model for exocytosis.

Binding of serotonin and dopamine to 'serotonin binding proteins' in bovine frontal cortex: evidence for iron-induced oxidative mechanisms.

Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.

Receptor-mediated internalization of angiotensin II in bovine adrenal medullary chromaffin cells in primary culture

Binding and internalization of angiotensin II (AII) were studied on bovine adrenal medullary cells in primary culture. Binding of [125I]AII was reversible, saturable, specific and showed high affinity. AII was found to be internalized by bovine adrenal medullary cells. Monensin increased whereas phenylarsine oxide (PhAsO) decreased the internalization. Excess of unlabelled AII or saralasin could block the internalization, indicating a receptor mediated internalization process. The kinetic analysis indicated that, during the first 4 min, about 25% of the membrane bound ligand was internalized per min and the recycling of internalized ligand and receptor initiated around 4 min.

The chromaffin granule — Plasma membrane interaction as a model for exocytosis: Quantitative release of the soluble granular content

Abstract Bovine adrenal medullary plasma membranes induce the release of soluble components from chromaffin granules in a Ca2+ dependent manner. This interaction can be modulated by changing the temperature. Correlation of the concentrations of four released soluble markers (catecholamines, soluble protein, ATP and dopamine-β-hydroxylase) in samples incubated at different temperatures revealed that those markers were liberated simultaneously. Their ratio did not differ significantly from the ratio measured in chromaffin granule lysates. These results suggest the release of the total granular content upon incubation with plasma membranes. Further analysis of the free catecholamines showed a preferential release of adrenalin.

Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS‐PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA‐immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA. these CGA‐processing products are released from the sheep spleen. The secretion of CGA‐derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.

Mass spectrometer characterization of bovine chromaffin granule peptides related to chromogranin B

Peptides were extracted from the lysate of isolated bovine chromaffin granules. Following reversed-phase HPLC purification, the fractions were analyzed by FAB/MS. The presence of methionine-enkephalin and leucine-enkephalin was indicated by their chromatographic retention time and by the m/z value of their protonated molecules. As to five new peptides related to chromogranin B, prominent protonated molecules were observed at m/z 1746, 1446, 1333, 977 and 901. Trypsinolysis resulted in a common loss of a component with mass 545, pointing to a structural relationship and a common precursor molecule. The peptide showing a (M+H)+ ion at m/z 1746 could be identified as a novel, recently reported, neuropeptide derived from chromogranin B, whereas the other peptides with (M+H)+ ions at m/z 1446, 1333, 977 and 901 could be characterized as smaller fragments of this peptide. Peptidase-guided sequence analysis and MS/MS analysis provided sequence information.

Conus venom interaction with α2-adrenergic receptors in calf retina membranes

?(2)-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [(3)H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the ?(2)-receptors with the following order of potency: C. planorbis (IC(50) = 2.1 ?g protein/ml) ? C. tessulatus (IC(50) = 2.7) >C. eburneus (IC(50) = 19) >C. textile (IC(50) = 54) >C. geographus (IC(50) = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( ? )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-?(2)-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of ?(2)-receptors in an antagonistic manner.

In vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules: Specific control by Ca2+

SummaryThe control of the in vitro interaction between bovine adrenal medullary plasma membernes and chromaffin granules by calcium has been studied. This interaction, which has previously been shown to result in the release of the soluble granular content, is a possible cell-free model for exocytosis. The plasma memberane-induced catecholamine release was stimulated when the [Ca2+] exceeded 2×10−7M. A maximal release was reached at 10−5M with a half maximal response around 10−6M. Mg2+ was not able to stimulate the system in the absence of Ca2+. These data suggest a high specificity of the calcium controlled exocytotic mechanism and the absence of an antagonism by high concentrations (10−5–10−1M) of magnesium at the exocytotic site itself.

Isolation and characterization of a cytosolic phospholipase A2 from bovine adrenal medulla.

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A2 (PLA2), which is localized in the cytosol. In the present study, this PLA2 was purified 1,097‐fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µM Ca2+ and has a pH optimum near 8.0. On sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA2 is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X‐100 (0.01%) and deoxycholate (1 mM) but inhibited at higher concentrations (0.1% and 3 mM, respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p‐Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA2, has little effect until 100 µM, and 2–10 mM dithiothreitol totally inactivated the enzyme. The purified PLA2 displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn‐2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA2, which was isolated and characterized in this study, represents a type of PLA2 that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA2 will help to reveal whether the enzyme is important for exocytosis.