Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.

We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.

Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells.

Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca(2+) and guanosine 5'-[gamma-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20-30 min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25 microM ATP can extend responsiveness significantly; this effect is maximal at 50 microM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPalpha and PITPbeta have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C(16)), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C(16) suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.

Thirty years of stimulus-secretion coupling: from Ca(2+) toGTP in the regulation of exocytosis.

Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognised as only one of a number of intracellular activators. In cells of haematopoietic origin (including mast cells), the key activator is one or more GTPases. Cells of this class, stimulated with GTPgammaS can undergo exocytosis in the effective absence of Ca(2+). A number of GTP-binding proteins that mediate exocytosis (G(E)) have been proposed but the best evidence supports roles for members of the Rho family of monomeric GTPases and for betagamma-subunits derived from G(i3). While preactivated Rac and Cdc42 can induce secretion from permeabilised mast cells in the absence of a guanine nucleotide betagamma-subunits only act to enhance the secretion induced by other GTP-binding proteins (likely to be members of the Rho family of monomeric GTPases). Further work is required to identify downstream effectors activated by these GTP-binding proteins and to show how they interact with the SNAP and SNARE isoforms known to be present in these cells.

Regulation of exocytosis from rat peritoneal mast cells by G protein beta gamma-subunits.

We applied G protein-derived beta gamma-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPgammaS over approximately 20-30 min. beta gamma-Subunits applied to the permeabilized cells retard this loss of sensitivity to stimulation (run-down) and it can be inferred that they interact with the regulatory mechanism for secretion. While alpha-subunits are without effect, beta gamma-subunits at concentrations >10(-8 )M enhance the secretion due to Ca2+ and GTPgammaS. Unlike the small GTPases Rac and Cdc42, beta gamma-subunits cannot induce secretion in the absence of an activating guanine nucleotide, and thus further GTP-binding proteins (likely to be Rho-related GTPases) must be involved. The enhancement due to beta gamma-subunits is mediated largely through interaction with pleckstrin homology (PH) domains. It remains manifest in the face of maximum activation by PMA and inhibition of PKC with the pseudosubstrate inhibitory peptide. Soluble peptides mimicking PH domains inhibit the secretion due to GTPgammaS and block the enhancement due to beta gamma-subunits. Our data suggest that beta gamma-subunits are components of the pathway of activation of secretion due to receptor-mimetic ligands such as mastoparan and compound 48/80.

Ontogeny of epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A in the adrenal gland of pigs.

OBJECTIVE: To obtain data on the ontogeny of catecholamines and other chromaffin vesicle components, which could serve as a basis for the study of their role during fetal life in normal and pathologic conditions. DESIGN: Epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A contents were measured in the porcine adrenal gland during various stages of gestation. ANIMALS: 934 porcine fetuses representing 22 gestational ages between 43 and 108 days. PROCEDURE: Total homogenates of adrenal glands were extracted and contents of different neurochemical markers were measured, using high-performance liquid chromatography, immunoassays, and western blotting. Immunohistochemical studies also were performed. RESULTS: Epinephrine and norepinephrine contents as a function of gestational age can be represented by a sigmoidal curve. Norepinephrine content rises early in gestation, whereas epinephrine content increases later. Maximal increase was significantly higher for epinephrine content. A progressive appearance of separate epinephrine- and norepinephrine-storing cells was documented. Dopamine-beta-hydroxylase content as a function of gestational age can be adequately represented by a parabolic curve. No quantitative changes in chromogranin A concentration were observed, but western blotting revealed qualitative changes with progressing gestational age. CONCLUSIONS: Important changes occur in catecholamine formation around day 60 of gestation. The sharp increase in epinephrine/norepinephrine contents and the appearance of separate epinephrine- and norepinephrine-storing cells may be related to the progressive splanchnic innervation of the adrenal gland. The presence of chromogranin A early in gestation may indicate its necessity for catecholamine storage.

Mononuclear-cell peptide mediation of chromaffin-cell epinephrine secretion.

Previously, we have shown that novel mononuclear-cell-derived factor(s) [molecular weight (MW) < 3,000] stimulate the release of epinephrine (EPI) from adrenal medullary chromaffin cells to levels comparable to that of maximal cholinergic stimulation. The present study provides evidence that the observed bioactivity is due to the action of a single peptide of 627 Da apparent MW. The peptide nature of the bioactive component was suggested by a decreased bioactivity after acid hydrolysis as well as altered bioactivity subsequent to peptidase (carboxypeptidase Y, leucine aminopeptidase) treatment. The bioactive conditioned-medium (CM) peptide(s) were isolated and further characterized using SDS-PAGE analysis. SDS-PAGE separation of G-25 Sephadex purified CM shows that bioactivity resides in a single peptide band. Additional studies revealed that CM also mediates norepinephrine release from sympathetic ganglia cells. Regulation of peptide production was shown to involve negative feedback in that incubation with mononuclear cells with EPI prevented further bioactive peptide release. This feedback inhibition was partially blocked by the beta-adrenergic receptor antagonist propranolol. These findings suggest a novel and potentially important mechanism by which the immune system can alter neuroendocrine function.

Umbilical cord dopamine beta-hydroxylase, chromogranin A and met-enkephalin after conditions associated with chronic intrauterine stress.

OBJECTIVE: To evaluate whether the markers of autonomic nervous system activity, dopamine beta-hydroxylase (DBH), chromogranin A (CGA) and met-enkephalin (E), are different in cord blood from neonates born after conditions associated with chronic intrauterine stress (CIUS) as compared to neonates born after a normal pregnancy. STUDY DESIGN: 61 newborns (median BW 2,840 g, range 617-4270 g) born after a pregnancy complicated by maternal hypertension, maternal smoking, maternal diabetes mellitus or intrauterine growth retardation (STR group) were compared with 88 neonates (median BW 2,910 g, range 4,00-4,370 g) who had not suffered from such intrauterine conditions. DBH, CGA and E were measured in the cord blood of both groups. RESULTS: When both groups were taken together, high DBH values were best related to maternal smoking (p = 0.004) and low E levels to maternal diabetes (p = 0.02). Within the STR group, high DBH values were best related with all conditions linked with CIUS (p = 0.008), E levels were best linked with the combination of intrauterine growth retardation (positive correlation) and maternal diabetes (negative correlation) (p = 0.03). For CGA there was only a weak positive relation with maternal smoking (p = 0.3). CONCLUSION: Certain intrauterine conditions associated with CIUS, especially maternal smoking, may lead to alterations of the autonomic nervous system as revealed by some of its markers in cord blood of neonates. This may be important in the pathogenesis of certain conditions after birth, such as the sudden infant death syndrome.

Fe(2+)-mediated binding of serotonin and dopamine to skeletal muscle actin: resemblance to serotonin binding proteins.

Fe2+ stimulates the binding of [3H]serotonin and [3H]dopamine to rabbit skeletal muscle actin. This binding is inhibited by reducing agents (sodium ascorbate, vitamin E), by superoxide dismutase and by sulfhydryl group-modifying reagents (N-ethyl-maleimide, 2,2'-dinitro-5,5'-dithiobenzoic acid). The effect of Fe2+ is mimicked by oxidants (sodium periodate, potassium nitroso-disulfonate) and by superoxide radicals. Once formed, the binding cannot be decreased by a large excess of monoamine. It is proposed that Fe2+ catalyses the autoxidation of the monoamines by generating oxygen free radicals, and the oxidation products are likely to bind covalently to exposed cysteine residues of actin. Digestion of [3H]dopamine-labelled actin by cyanogen bromide and then by V8 protease (EC3.4.21.19) yields two labelled peptides whose apparent molecular weights (4.1 and 1.2 kDa) are compatible with the labelling of cysteine-10 and -374. Fe2+ also inactivates some of the binding sites on actin. This inactivation, and the covalent nature of the binding precludes the interpretation of monoamine saturation and competition binding data in terms of reversible bimolecular interactions.

Interaction of serotonin- and dopamine-related neurotoxins with "serotonin binding proteins" in bovine frontal cortex.

Binding of [3H]serotonin and [3H]dopamine to serotonin-binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+. This group recently attributed this effect of Fe2+ to its ability to enhance the oxidation of [3H]serotonin and [3H]dopamine in the presence of dissolved molecular oxygen, and to the ability of the formed oxidation products to bind covalently to cysteine residues of SBP. In this study it is shown that the binding of both ligands is potently inhibited by dopamine as well as by several catecholamine-and serotonin-related neurotoxins: adrenochrome, 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 6-hydroxydopamine and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. In contrast, serotonin can only potently inhibit part (36%) of the [3H]dopamine binding, while 1,2,3,4-tetrahydroisoquinoline is only a weak competitor for both ligands. Potent inhibition by the toxins is associated with the presence of electrophilic centres at the aromatic ring, either of the products themselves (adrenochrome) or of their oxidation products (all other competitors). These findings suggest that "SBP" represent an important target for the Fe(2+)-mediated binding of [3H]-serotonin, [3H]dopamine and related neurotoxins.

Soluble serotonin and catecholamine binding proteins in the bovine adrenal medulla.

The soluble serotonin-binding proteins (SBP) present in the adrenal medulla and in chromaffin cells, are very similar to those reported for the bovine brain and retina. Binding of [3H]serotonin and [3H]dopamine to these SBP is increased by Fe2+ but not by Fe3+. At an optimal concentration of Fe2+ (0.1 mM) these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 124 +/- 28 pmol/mg protein, KD = 0.51 +/- 0.13 microM) and [3H]dopamine (Bmax = 685 +/- 118 pmol/mg protein, KD = 0.46 +/- 0.06 microM). Binding of [3H]dopamine is also increased by Cu2+ and Mn2+, but to a lesser extent than by Fe2+. Catecholamines are good competitors for [3H]serotonin binding (Ki = 0.31 microM for dopamine, 0.6 microM for adrenaline and 0.9 microM for noradrenaline). The serotonin binding proteins from adrenal medulla elute in the void volume of a Sephacryl 100 HR gel filtration column, reflecting aggregation, and migrate mainly with an apparent molecular weight of 45 kDa in native polyacrylamide gel electrophoresis experiments. Subcellular localization studies and release experiments suggest that SBP are not present in chromaffin granules, but in the cytosol of purified chromaffin cells. The present data suggest that these proteins must have other functions than storing monoamines in synaptic vesicles.

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.

Serotonin binding proteins in bovine retina: binding of serotonin and catecholamines.

Serotonin binding proteins (SBP) are present in the soluble fraction of bovine retina homogenates. These proteins can be precipitated with 30% ammonium sulphate and their binding and physicochemical characteristics are very similar to those of SBP in bovine and rat brain. Binding of [3H]serotonin to bovine retina SBP requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 242 +/- 10 pmol/mg protein, KD = 0.22 +/- 0.44 microM). Competition binding studies reveal that serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are potent competitors (K1 from 0.12 to 0.3 microM). In both cases, the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine SBP interactions can also be demonstrated directly by binding experiments with [3H]dopamine. Binding of this catecholamine is greatly enhanced by Fe2+, to a lesser extent by Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component is saturable (Bmax = 505 +/- 30 pmol/mg protein. KD = 0.34 +/- 0.04 microM). The SBP from bovine retina show the same physicochemical properties as SBP from bovine and rat brain: they elute immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and they migrate with apparent molecular weights of respectively 43 kDa and 57 kDa on native polyacrylamide gel electrophoresis. The serotonin-storing role of SBP in serotonergic neurones has already been well documented.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin-binding proteins in the bovine cerebral cortex: interaction with serotonin and catecholamines.

The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [3H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [3H]serotonin binding (Bmax = 120 +/- 12 pmol/mg protein, KD = 0.12 +/- 0.04 microM, n = 3). Binding of [3H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (Ki from 0.1 to 0.3 microM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [3H]dopamine is greatly enhanced by Fe2+, Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component of [3H]dopamine is saturable (Bmax = 279 +/- 64 pmol/mg protein, KD = 0.19 +/- 0.02 microM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57-58 kDa on native polyacrylamide gel electrophoresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.