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J D Vincent

Publications and source records attributed to J D Vincent.

At least 55 records · Page 3Linked to original sources
PubMed1996

Neurotrophin-3 increases intracellular calcium in a rat insulin-secreting cell line through its action on a functional TrkC receptor.

Pancreatic beta cells and neuronal cells show a large number of similarities. For example, functional receptors for nerve growth factor are present in beta cells. Here we investigate whether TrkC, a neuronal high affinity receptor for neurotrophin-3, is expressed in the insulin-secreting cell line INS-1. We demonstrate the expression in INS-1 cells of mRNAs coding for TrkC identical in size to those found in the brain. As in neuronal cells, different alternatively spliced forms of TrkC mRNA, differing by the insertion of an alternative exon in their kinase domain, were expressed in INS-1 cells. TrkC protein is also expressed in INS-1 cells and is functional. Indeed, when INS-1 cells were treated with neurotrophin-3, TrkC became phosphorylated on tyrosine residues, and the expression of early response genes was induced. This activation of the receptor was paralleled by a rapid and transient increase in cytosolic free calcium due to an influx of extracellular calcium. Functional receptors for NT-3 are thus expressed in INS-1 cells. This cell line provides a new model for the study of NT-3 signal transduction and should be useful in the understanding of the role of neurotrophins in insulin-secreting cells.

Amino Acid Sequence↗
PubMed1996

Expression of a novel form of the p56lck protooncogene in rat cerebellar granular neurons.

The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti-lck antibody, an lck-like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56-kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56-kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription-PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5' end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120 to neurons.

3T3 Cells↗
PubMed1996

[From phylogenesis to physiology: dopamine receptors].

Communication between neurones requires specialised proteins called receptors which fix the neuromediator (e.g. dopamine) and transmit a message to the cell interior. For a given neuromediator there exist several types and subtypes of receptors, which make up multigenic families. These gene families have come to be thanks to successive duplications of one initial gene in the course of evolution. Thus dopamine receptors of the D1 class include up to four subtypes in vertebrates. The cloning and subsequent study of D1 genes in a sample of species representative of the major evolutionary stages in vertebrates has led to the localisation of the duplication phases of the genes at the origin of the functional diversity of these receptors.

Animals↗
PubMed1995

HIV-1 envelope proteins gp120 and gp160 potentiate NMDA-induced [Ca2+]i increase, alter [Ca2+]i homeostasis and induce neurotoxicity in human embryonic neurons.

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N-methyl-D-aspartate (NMDA)-induced current were recorded from neurons maintained in culture for 10-30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca2+ concentration ([Ca2+])i, gp120 and gp160 (20-250 pM) potentiated the large rises in [Ca2+]i induced by 50 microM NMDA. The potentiation of NMDA-induced Ca2+ responses required the presence of Ca2+ in the medium, and was abolished by the NMDA antagonist D-2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca2+ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20-250 pM gp120 or gp160 resulted in an increase in [Ca2+]i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca2+]i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immunofluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and

Calcium↗
PubMed1995

Dopamine D1 receptor modulates the voltage-gated sodium current in rat striatal neurones through a protein kinase A.

1. Whole-cell recordings were made from striatal neurones obtained from neonatal rats and maintained in primary cultures. The effects of dopamine D1 receptor activation were studied on the voltage-gated sodium current. 2. Bath application of a specific D1 agonist, SKF38393 (1 microM), reduced the neuronal excitability recorded in current-clamp by increasing the threshold for generation of action potentials. 3. In voltage-clamp recordings, SKF38393 (1 microM) reversibly reduced the peak amplitude of the sodium current by 37.8 +/- 4.95%. This effect was reversed by the D1 antagonist SCH23390 and was blocked by the intracellular loading of GDP-beta-S (2 mM) suggesting GTP-binding protein involvement. 4. The D1 agonist reduced the peak amplitude of the sodium current without significantly affecting (i) the voltage dependence of the current-voltage relationship, (ii) the voltage dependence of the steady-state activation and inactivation, (iii) the kinetics of the time-dependent inactivation, and (iv) the kinetics of recovery from inactivation. 5. The peak amplitude of the sodium current was progressively reduced by intracellular loading of cyclic AMP-dependent protein kinase (100 U ml-1). 6. Diffusion of a specific peptide inhibitor of the cyclic AMP-dependent protein kinase (PKI; 10 microM) into the cytosol of neurones blocked the effect of the D1 agonist on the sodium current amplitude. 7. These results demonstrate that dopamine acting at the D1 receptor reduces the amplitude of the sodium current without modifying its voltage- and time-dependent properties. This effect involves activation of the cyclic AMP-dependent protein kinase and results in a depression of the striatal neuronal excitability by increasing the threshold for generation of action potentials.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1995

Reduction of dopamine D2 receptor transduction by activation of adenosine A2a receptors in stably A2a/D2 (long-form) receptor co-transfected mouse fibroblast cell lines: studies on intracellular calcium levels.

A stably co-transfected mouse fibroblast cell line, which expresses the long form of the human dopamine D2 receptor and the dog adenosine A2a receptor, was used to analyse the mechanism underlying changes in the cytosolic free calcium concentration ([Ca2+]i) induced by activation of D2 (long-form) receptors and its modulation by the A2a receptor agonist CGS 21680 by means of fura-2 imaging. Quinpirole (1-1000 nM), a D2 receptor agonist, caused a concentration-dependent increase in [Ca2+]i. Haloperidol, a preferential D2 receptor antagonist, completely blocked this [Ca2+]i response to quinpirole. Preincubation with Ca(2+)-free medium containing 2 mM EGTA or a medium containing 320 mM ethanol, an inositol 1,4,5-triphosphate receptor antagonist, substantially diminished the increase in [Ca2+]i evoked by quinpirole. Furthermore, quinpirole totally failed to elevate [Ca2+]i in a medium containing both 2 mM EGTA and 320 mM ethanol. CGS 21680 (1-500 nM) did not, by itself, exert any significant effect on [Ca2+]i. However, 100 nM of CGS 21680 substantially counteracted the [Ca2+]i responses to quinpirole (10-1000 nM). Moreover, this counteraction still occurred after blocking Ca2+ mobilization from intracellular stores with ethanol, but disappeared when the cells were pretreated with the Ca(2+)-free medium containing 2 mM EGTA. Our findings imply that the D2 (long-form) receptors in the present fibroblast cell line can raise [Ca2+]i both via Ca2+ influx from the extracellular medium and Ca2+ release from intracellular stores via activation of inositol 1,4,5-triphosphate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1995

Sex steroid hormones change the differential distribution of the isoforms of the D2 dopamine receptor messenger RNA in the rat brain.

The two isoforms of the rat dopamine D2 receptor are generated by alternative splicing of the pre-messenger RNA and differ in the length of their third cytoplasmic loop involved in coupling to G-proteins. As quantified by polymerase chain reaction, the long isoform D2L is predominant in the pituitary gland, the striatum and to a lesser extend in the olfactory tubercle, whereas the short isoform D2S is relatively more abundant in the hypothalamus and the substantia nigra. Changes in circulating sex hormone levels modulated the splicing without affecting the total amount of D2 receptor messenger RNA. Castration of male rats increased the ratio D2L/D2S in the pituitary, hypothalamus and substantia nigra, and decreased it in the olfactory tubercle. Testosterone substitution reversed the effect of castration in the pituitary and olfactory tubercle but not in the substantia nigra. In castrated rats, 17beta-estradiol had a similar effect to that of testosterone in the olfactory tubercle, indicating that testosterone may act after aromatization of estradiol. In the hypothalamus, 17beta-estradiol alone reversed the effect of castration. In the striatum, neither castration nor hormonal treatments modified the splicing of the D2 receptor mRNA. Treatment of animals with specific androgen and estrogen receptor blockers confirmed that steroids were acting through their specific intracellular receptors. These observations suggest a molecular mechanism, physiologically relevant, by which circulating sex hormones could modulate dopamine transmission in areas implicated in reproductive and parental behaviours.

Animals↗
PubMed1995

An evolutionary view of drug-receptor interaction: the bioamine receptor family.

The large molecular diversity of receptors and their subtypes means that the pharmacologist is faced with many puzzling characterization questions. First, the molecular diversity of the receptors is deciphered only in part by a pharmacological approach, which precludes a satisfactory receptor classification based solely on pharmacological characteristics. Second, the physiological counterpart of the numerous subtypes of receptors specifically activated by single endogenous ligands remains unclear. Here, Philippe Vernier and colleagues use the example of the bioamine G protein-coupled receptors to show that many of the apparent inconsistencies that emerge from pharmacological and molecular characterizations of receptors can be better understood if the evolutionary history of the receptors is taken into account.

Amines↗
PubMed1994

[Biology of pleasure].

Pleasure is not an "extra", or bonus bringing a little more soul to certain of our acts; it is a fundamental part of animal life. It is just as difficult to define as spirit, but nonetheless man is very conscious of it, and it is found to be associated with certain animal behaviours. The study of the biology of pleasure entails 1) showing how it intervenes in relation with "need" in the regulation of major homeostatic functions, 2) studying its participation in the opponent processes which determine given behaviours, 3) categorising the hormones and neurotransmitters it brings into play, 4) determining its presence in drug addiction and pathological consommatory behaviour.

Animals↗
PubMed1994

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Adrenal Cortex↗
PubMed1994

Rab3 proteins: key players in the control of exocytosis.

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Animals↗
PubMed1994

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Amino Acid Sequence↗
PubMed1993

Inhibition of Rab3B expression attenuates Ca(2+)-dependent exocytosis in rat anterior pituitary cells.

Low-molecular-weight GTP-binding proteins (small G proteins) of the Rab family have been proposed to act as central regulators of vesicular traffic, and proteins of the Rab3 subfamily (Rab3A, B, C and D) are thought to be associated with membrane vesicles or granules undergoing exocytotic fusion with the plasma membrane. Rab3A is highly expressed in brain, whereas Rab3B is the major form found in rat anterior pituitary gland. We report here that antisense oligonucleotides against Rab3B, introduced into pituitary cells using the whole-cell patch clamp technique, specifically and reversibly block expression of Rab3B. We find that calcium-dependent exocytosis is inhibited without affecting endocytosis. Antisense oligonucleotides directed against Rab3A have no effect. Our results indicate that Rab3B is likely to be a key intracellular signalling molecule which can control exocytosis downstream of other calcium-dependent processes in anterior pituitary cells.

Animals↗
PubMed1993

Alterations in vasopressin and oxytocin messenger RNA in the rat supraoptic nucleus during dehydration-rehydration evaluated by in situ hybridization and northern blotting.

Male rats were deprived of water for 5 days, and then given water ad libitum for 3, 7, 10 or 14 days. Plasma osmolarity returned to normal in less than 3 days, while pituitary vasopressin (AVP) and oxytocin (OXT) only returned to control levels after 14 days. Sections of the supraoptic nucleus (SON) were hybridized with 35S-labelled cDNA (OXT) or oligonucleotide (AVP) probes. Relative AVP and OXT mRNA contents were quantitated by counting the number of silver grains on a large standard area of the SON, then extrapolating this value to the volume of the whole SON (deduced from surface areas of all the sections). Dehydration significantly enlarged the volume of the SON (x 1.54) and increased the AVP and OXT mRNAcontent (x 2). During rehydration, both SON volume and density of silver grains were higher than normal for at least 7-10 days, although levels started to fall by day 3. The distribution of individual cells according to their silver grain densities remained unimodal during the dehydration-rehydration sequence with an extension, then a return to normal of the distribution range. Maximum sizes of AVP and OXT mRNAs on Northern blots of RNAs extracted from 5 pooled SONs were observed on dehydration day 5. The size of these species fell progressively, reaching control values by rehydration day 14. We conclude that during rehydration, at a time when most of the putative inducers of gene transcription are no longer activated, the peptidergic deficit was accompanied by an increased level of AVP and OXT mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Hormonal control of neural function in the adult brain.

Although the mechanisms by which peripheral hormones modulate complex behaviors are far from being well understood, recent advances in deciphering the mechanisms of hormone action in the brain are promising. Current areas of interest include the molecular mechanisms of steroid receptor action, the steroid modulation of synaptic function, and the mediation of steroid-regulated neuronal and glial plasticity by growth factors or proteins associated with brain development.

Adult↗
PubMed1993

Mechanism of the prolactin rebound after dopamine withdrawal in rat pituitary cells.

To study the mechanism underlying the effect of dopamine withdrawal on prolactin release, continuous perfusion experiments were performed on rat lactotroph-enriched primary cultures. Removal of dopamine (10(-7) M) after a short-term application (15 min) produced a rebound of prolactin secretion, which was enhanced by pretreatment of the cell culture with 17 beta-estradiol (10(-8) M for 48 h). Ca2+ channel blockade by Co2+ (1 mM) abolished the rebound in prolactin release. An increase in intracellular adenosine 3',5'-cyclic monophosphate by either forskolin (5 microM) or 3-isobutyl-1-methylxanthine (100 microM) enhanced the prolactin rebound after dopamine withdrawal. Application of thyrotropin-releasing hormone (10(-7) M) increased the prolactin rebound after dopamine withdrawal with a maximum effect obtained by commencing treatment immediately after removal of dopamine. Pretreatment of cell cultures with pertussis toxin (100 ng/ml, for 10 h) totally abolished the effects of dopamine on prolactin secretion. The dopamine agonist bromocriptine (10(-9) M) significantly decreased prolactin secretion, but no rebound effect was observed after its removal. We conclude that the rebound of prolactin release after dopamine treatment involves the influx of Ca2+.

Animals↗