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B A Demeneix

Publications and source records attributed to B A Demeneix.

At least 55 records · Page 3Linked to original sources
PubMed1991

Gene transfer into intact vertebrate embryos.

Intact chick embryos at 40 h incubation were transfected in vivo with chimeric vectors expressing chloramphenicol acetyl transferase (CAT) under different promoter sequences. The cationic lipid, dioctadecylamidoglycyl spermine (DOGS) used as the transfecting agent had no noticeable toxic effects on embryonic development. CAT activity was monitored 48 h post-transfection on homogenates of embryos dissected free of all annexes. Of the various constructs tested, those containing the AP-1 response element linked to CAT (TRE-tk-CAT) gave high expression and consistent enzyme responses within groups. Co-transfection experiments in which embryos were exposed simultaneously to a CAT vector containing the cAMP response element and to a vector expressing the catalytic subunit of protein kinase A showed that the promoters of the introduced genes can be regulated by their respective transacting factors. This method may therefore represent a general tool for introducing genes into intact vertebrate embryos at precise developmental times.

Animals↗
PubMed1990

Differentiation to a neuronal phenotype in bovine chromaffin cells is repressed by protein kinase C and is not dependent on c-fos oncoproteins.

We investigated the intracellular signals underlying the neurotrophic response of adult bovine chromaffin cells to histamine and basic fibroblast growth factor (bFGF). Histamine produced significant neurite outgrowth within 48 hr, whereas the response to bFGF developed after 1 week. H7, a protein kinase C (PKC) inhibitor potentiated both the histamine and the bFGF responses, while another PKC antagonist, staurosporine, induced a rapid and efficient differentiation response when applied alone. These observations suggest that basal PKC activity is required for stabilization of the endocrine phenotype in these cells. They contrast with findings on NGF induction of neurite outgrowth in PC12 cells where PKC promotes differentiation, apparently by activating the fos/jun complex. Thus, we examined the role of c-fos in our model. Both histamine and bFGF induced c-fos gene expression transiently. To determine whether increased levels of c-fos oncoprotein were essential to the differentiation process, we used a hybrid arrest approach employing an innovative transfection technique applicable to primary culture systems. Transfection with plasmid pSVsof, producing antisense c-fos mRNA, reduced c-fos oncoprotein levels but did not diminish histamine-induced neurite outgrowth. We infer that histamine-induced differentiation in bovine chromaffin cells is independent of increased levels of c-fos oncoprotein.

Adrenal Medulla↗
PubMed1989

Thyrotropin-releasing hormone stimulates porcine melanotrope cells in primary culture.

The action of thyrotropin-releasing hormone (TRH) on melanotrope cells maintained in primary culture was studied with biochemical and electrophysiological techniques. TRH effects on polyphosphoinositide (PPI) breakdown was measured in [3H]myoinositol labelled cells maintained in suspension for 24 hours or in primary culture. TRH (50 nM) or its potent analogue (3Me-His2)-TRH increased total PPI levels by 50-125% in separate experiments after 30 min of treatment whereas corticotropin-releasing hormone (CRF) was without effect. The effect of TRH was dose-dependent (ED50 = 5 nM), the maximal effect being reached with 50 nM TRH. Using the patch-clamp technique in the cell-attached configuration spikes were recorded extracellularly. In 6 of the 13 cells tested, (3Me-His2)-TRH (10 nM) elicited an increase in the spontaneous spiking rate. Furthermore, TRH (50 nM) increased melanocyte-stimulating hormone (alpha-MSH) secretion 2-fold after 8 h of treatment. These results suggested that TRH activated phospholipase C and electrical activity in melanotrope cells; the resulting phosphoinositide breakdown and increase in intracellular free Ca2+ ultimately led to a stimulation of hormone release.

Action Potentials↗
PubMed1989

Ca2+ regulates hormone secretion and proopiomelanocortin gene expression in melanotrope cells via the calmodulin and the protein kinase C pathways.

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Spontaneous spiking activities of porcine pars intermedia cells: effects of thyrotropin-releasing hormone.

The electrophysiological properties of porcine melanotrophs in primary culture were studied with patch clamp techniques. In the cell-attached (c/a) configuration, extracellular spikes were recorded but in only 47 of the 259 cells examined; active cells were more frequently found in short-term cultures (48 h, 22 out of 71). In the whole-cell (WCR) configuration, the mean resting potential was -46.6 mV and the mean input resistance 2.41 G omega. In the current clamp mode, of the 99 cells recorded, 39.4% displayed spontaneous spiking activities, 14.1% spiked when a depolarizing current was applied, and 46.5% were silent. Two patterns of spontaneous activity were recorded. The first consisted of rapid membrane depolarizations (mean duration: 16.0 ms) which reached a mean value of +8.9 mV. These action potentials occurred either at random intervals or at a mean frequency of 1.19 Hz. Tetrodotoxin (TTX), a sodium channel blocker, completely abolished these action potentials. The second pattern consisted of regular bursts (mean duration: 1.69 s and mean frequency: 0.15 Hz) of spikes with smaller amplitudes (culminating at -9.5 mV) and greater durations (79.7 ms). This pattern could be recorded in the presence of TTX in the external medium. In the c/a configuration, thyrotropin-releasing hormone (TRH) and (3Me-His2)-TRH enhanced spike frequency, whereas histidyl-proline-diketopiperazine, a metabolite of TRH, had no effect. In WCR, out of the 27 cells tested, TRH (5.10(-9)-5.10(-8) M) induced firing in 4 quiescent cells and increased the frequency of action potentials in 4 spontaneously active cells. This was usually, but not necessarily, preceded by a hyperpolarization (n = 5). TRH (5.10(-8) M) enhanced the secretion of melanocyte-stimulating hormone (alpha MSH) from perfused isolated melanotrophs by 62.8 +/- 9.8% (n = 9) over basal levels. (3Me-His2)-TRH and (Pro3)-TRH mimicked the TRH response, whereas histidyl-proline-diketopiperazine was without effect.

Action Potentials↗
PubMed1988

Dopamine inhibition of proopiomelanocortin gene expression in the intermediate lobe of the pituitary. Interactions with corticotropin-releasing factor and the beta-adrenergic receptors and the adenylate cyclase system.

The effects of dopamine on proopiomelanocortin (POMC) gene expression were compared in primary cultures of the anterior and intermediate lobes of the rat pituitary. A single-stranded POMC complementary DNA was used to quantitate POMC messenger RNA levels. Treatment with dopamine (1 microM) for 48 h reduced POMC messenger RNA levels in the intermediate lobe by 77%, but had no effect on POMC gene expression in the anterior lobe. Dopamine D2 receptors were implicated in the response, as bromocriptine (100 nM). reproduced the dopamine inhibition. The responses to dopamine and bromocriptine were antagonized by haloperidol (10 microM). The decrease in POMC messenger RNA levels was dose dependent with ED50 values of about 50 and 0.1 nM for dopamine and bromocriptine, respectively. The accumulation of POMC-derived peptides, beta-endorphin and alpha-melanocyte-stimulating hormone, over 2 days was measured by radioimmunoassay and was shown to parallel the changes in POMC synthesis. The dopamine-induced inhibition of intermediate lobe POMC synthesis was unaffected by isoprenalin (5 microM) and corticotropin-releasing factor (10 nM), although these treatments had stimulatory effects when tested alone. Activating adenylate cyclase with forskolin (1 microM) or treatment with 8-bromocyclic adenosine monophosphate (1 mM) doubled POMC messenger RNA levels, and, when tested against these stimuli, bromocriptine still produced a 30% inhibition of POMC gene expression. These observations suggest that D2 receptor induced inhibition of POMC gene expression is not only mediated by a decrease in cyclic adenosine monophosphate levels. When cells were pretreated with pertussis toxin (100 ng/ml), the bromocriptine-induced inhibition was almost completely lost, suggesting that the dopaminergic inhibition is mediated by guanosine triphosphate binding proteins.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1-4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10(-8) M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I greater than Br greater than Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23-31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR42641, 1 microM) blocked activity evoked by GABA (1-10 microM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 microM, and were practically abolished at Cai 1 microM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 microM.

Animals↗
PubMed1986

Characterization of calcium and sodium currents in porcine pars intermedia cells.

The whole-cell configuration of the patch-clamp technique was applied to porcine pars intermedia cells. A tetrodotoxin-sensitive sodium current was recorded. Three types of calcium current were observed. Depolarizations from a holding potential of 100 mV elicited a transient current (ICaT), whereas depolarizations from a holding potential of -40 mV evoked a sustained current (ICaS). A third current (ICaN; N for neither) was activated by strong depolarization to +10 or +20 mV from holding potentials of - 100 mV. Increasing internal Ca2+ significantly reduced the amplitude of ICaS.

Animals↗
PubMed1986

GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release.

A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte-stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed.

Animals↗
PubMed1986

Ionic conductances related to GABA action on secretory and biosynthetic activity of pars intermedia cells.

We have examined the action of GABA on the electrical, secretory and synthetic activities of rat and porcine intermediate lobe (IL) cells in primary culture. Chloride and calcium currents were investigated using patch-clamp techniques. A chloride current activated by 1-100 microM isoguvacine, a specific GABA-A agonist, and antagonised by bicuculline and SR 95103 was recorded at the whole cell and single channel level current. Whole cell calcium currents were investigated and shown to be reduced by 40 microM cadmium, zero external calcium and 10 microM baclofen, a specific GABA-B receptor agonist. Both GABA-B receptor activation and use of calcium deficient medium inhibited peptide release from IL cells. Finally, pro-opiomelanocortin (POMC) mRNA levels were measured using a hybridization technique. Removal of calcium from the culture medium or long-term (48 hr) incubation with 10 microM GABA or muscimol (a mixed GABA-A and GABA-B agonist) significantly reduced POMC mRNA levels.

Animals↗
PubMed1986

[Electrophysiological and biochemical studies of GABA receptors in a primary cell culture of the pars intermedia of the porcine pituitary].

Using a primary culture of intermediate lobe (IL) we are investigating how GABA modulates stimulus-secretion coupling in the hypophysis. Two classes of GABA receptors have been described: GABA-A and GABA-B. We have characterised the GABA-A receptor using the patch clamp technique (Whole Cell Recording). GABA (10-100 microM) and isoguvacine (50 microM) a specific GABA-A agonist elicit an inward current with an equilibrium potential that coincides with Ecl-, this could explain the depolarising action of GABA-A agonists. When applying baclofen, the specific GABA-B agonist, no modifications of membrane potential were seen. Perfusion studies on alpha MSH release showed GABA (50 microM) and isoguvacine (50 microM) to potentiate Ba++-evoked secretion. The effects were antagonised by bicuculline and SR 95103, confirming GABA-A receptor action. Baclofen stereospecifically inhibited both basal and Ba++-evoked release. Together the results suggest the co-existence of the two classes of GABA receptors on the endocrine cells of the IL.

Animals↗
PubMed1984

Properties of the GABA receptors located on spinal primary afferent neurones and hypophyseal neuroendocrine cells of the rat.

Electrophysiological techniques have been used to study the pharmacological characteristics of GABA receptors in two in vitro preparations likely to provide the ionic basis for GABAergic inhibition of excitation-secretion coupling. The shortening of Ca2+ spikes duration by GABAB receptors was shown to occur in slow conducting dorsal root ganglion cells, independently of marked depression of inward calcium currents. Ion-selective electrodes (K+ or Ca2+) were used to show the presence of both GABAA and GABAB receptors on the neurosecretory terminals and gland cells from hypophyseal neuro-intermediate lobe (NIL). In this latter preparation, potentiation of hormone release was observed under GABAA receptor activation, whilst inhibition was seen with GABAB agonists.

Afferent Pathways↗
PubMed1984

Dual population of GABAA and GABAB receptors in rat pars intermedia demonstrated by release of alpha MSH caused by barium ions.

We have studied the effects of selective GABAA and GABAB agonists on alpha-melanophore stimulating hormone (alpha MSH) release from intact rat neurointermediate lobes (NIL) in vitro. Agonist effects were tested against either basal alpha MSH output or BaCl2 (5 mM)-evoked release. GABA (50 microM) produced a biphasic effect on basal release, with an enhancement followed by inhibition of release. The enhancement but not the inhibition was blocked by bicuculline methiodide (100 microM). Baclofen (10 microM), a specific GABAB agonist, reduced the basal and Ba2+-evoked hormonal release in a stereospecific manner. (-)-Baclofen (5 microM) was active whereas the (+)-isomer was inactive at the same concentration. Isoguvacine (50 microM) a specific GABAA agonist, potentiated the Ba2+-evoked release of alpha MSH. GABA (50 microM) mimicked this effect, and its action was antagonized by bicuculline methiodide (200 microM). The results suggest that both GABAA and GABAB receptors are present on the endocrine cells of the intermediate lobe.

Animals↗
PubMed1983

Thyroidal iodine and thyroglobulin in hibernating and active Richardson's ground squirrels Spermophilus richardsoni.

Thyroidal iodine, protein and thyroglobulin (TG) were investigated in a hibernator, Spermophilus richardsoni, sampled in the field and from laboratory-held winter colonies. In field animals, thyroidal iodine and protein contents were similar at onset of hibernation and at terminal arousal. Iodine content was increased in laboratory-held animals, a function of alimentary supply, but no differences were observed between hibernators and non-hibernators. Density sucrose gradients showed that approx. 20% of the TG was present as the 12S precursor sub-unit. No variations in the iodine content, nor the iodoamino acid composition of the TG occurred as a function of hibernation.

Amino Acids↗
PubMed1983

Electrophysiological study of GABAA versus GABAB receptors on excitation-secretion coupling.

Inwardly-directed Ca++-currents are caused by numerous types of action potentials which would not otherwise cause secretion. This process is regulated electrically and by neurotransmitters. We have studied in vitro the ionic mechanisms of GABA-mediated presynaptic inhibition and thereby the distinctive characteristics of GABAA and GABAB receptors: i.e., the GABAA system, which produces such short-lasting changes that there is an instantaneous reduction of spike amplitudes, in particular by opening Cl- -conductance, and the GABAB system, which results directly in inhibition of secretion due to a tonic depression of Ca++-currents. The principal aim was to determine if GABAB/Ca++ receptors could coexist on a membrane already possessing a large number of GABAA/Cl- sites available for presynaptic inhibition. Intracellular recordings of A delta and C dorsal root ganglion cell bodies were used as a model for the study of preterminal axonal membranes. Results were tentatively correlated with those obtained extracellularly by recording Ca++ and K+ movements (Cl- being assessed indirectly) from a set of other cells which also secrete neuropeptides by exocytosis: e.g., endings of unmyelinated fibres in the neurohypophysis and clusters of innervated gland cells in the pars intermedia. In recent years there has been a growing interest in receptors for neurotransmitters (e.g., monoamines, peptides, gamma-aminobutyric acid) modulating Ca++-dependent secretion in various biological systems (14-16, 24, 25, 29, 34). Modulation is possible either by changing the basic characteristics of the ionic currents during a standard action potential (Na+/K+ voltage transients being markedly altered by repolarizing K+ currents) or by any kind of direct action on voltage-dependent Ca++-channels: the latter will allow Ca++ to enter the cell in graded amounts not only as parts of well-defined Ca++-spikes (16, 14, 39; see also 24, 26, 28) but of well-defined Ca++-spikes (16, 14, 39; see also 24, 26, 28) but possibly also along complex sequences of tail-currents (for the biophysics, see 28, 29). Accordingly, it is essential to study presynaptic actions of transmitters in systems where spikes can be recorded, though presynaptic receptor activation can sometimes be identified by other means than spiking patterns (e.g., membrane effects in relation to other well-defined ionophores). The study of cells which are in contact with synapses synthesizing and releasing gamma-aminobutyric acid (GABA) has drawn much of the attention given to these problems.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed1982

Electrophysiological study with K+- and Ca2+-sensitive micropipettes of GABA receptors in the rat neurointermediate lobe in vitro.

An efflux of K+ and a decrease in extracellular Ca2+ activity are to be expected when GABA markedly depolarizes the membrane of unmyelinated axons or secretory cells. Accordingly, we used extracellular recordings of ionic movements to specify GABA receptor presence in parts of the pituitary having GABAergic innervation: the neurohypophysis and intermediate lobe (NIL). We identified a site of action having the same desensitization characteristics and pharmacological criteria as the GABA A receptor which modulates Cl- -conductance. Baclofen, a GABA B agonist, was without effect. The possible distribution of receptors and role of GABAergic synapses in modulating neurotransmitter and hormone release by the NIL are discussed.

Animals↗