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J D Vincent

Publications and source records attributed to J D Vincent.

At least 19 recordsLinked to original sources
PubMed1992

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Adenosine Diphosphate Ribose↗
PubMed1992

Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors.

This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Chronic stimulation of D2 dopamine receptors specifically inhibits calcium but not potassium currents in rat lactotrophs.

The present study examines the effect of chronic dopamine treatment, known to inhibit prolactin release from anterior pituitary, on two Ca2+ and K+ currents in cultured rat lactotrophs. K+ and Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. The two types of voltage-dependent Ca2+ currents are called SD and FD (slowly deactivating and fast deactivating current component, respectively) and the two types of voltage-dependent K+ currents, IA and IK. All current types were isolated by tail current analysis. The amplitude of both normalized calcium components depended on the length of the culture (n = 48) while normalized amplitudes of both potassium currents remained constant (n = 9). Incubation of cells during 72 h with 50 microM of Actinomycin D, an inhibitor of mRNA synthesis, suggested that this increase in Ca2+ currents involved the synthesis of proteins. Long-lasting D2 receptor stimulation (8 days; 10 nM RU 24213) prevented this selective effect through activation of a pertussis toxin-sensitive G protein. We also examined whether cyclic adenosine-3',5'-cyclic-monophosphate (cyclic AMP) or Ca2+/phospholipid-dependent protein kinase (protein kinase C) could affect this development of channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Efficiency of transcranial electrostimulation on anxiety and insomnia symptoms during a washout period in depressed patients. A double-blind study.

In order to test the efficacy of cerebral electrostimulation (electrosleep) as an alternative to drug therapy for the treatment of anxiety and insomnia, we conducted a double-blind study in a sample of 21 depressed inpatients submitted to a 5-day period of drug washout on admission to the psychiatric department. During this withdrawal period, anxiety and insomnia were exacerbated in the placebo group, whereas anxiety decreased and sleep duration improved in the active treatment group, with a divergent evolution during the 5-day washout period. The depressive criteria did not respond differentially to treatment, however. Thus, the effects of this drug washout period are markedly attenuated by cerebral electrostimulation, which is of possible interest in the management of psychotropic drug withdrawal.

Adult↗
PubMed1991

Maturation of a transient outward potassium current in mouse fetal hypothalamic neurons in culture.

The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (greater than six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of-20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (-80 mV) than that required for evoking the delayed rectifier potassium current (-40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation. The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.

Animals↗
PubMed1991

Physiological characterization of two functional states in subpopulations of prolactin cells from lactating rats.

1. Lactotroph cells from lactating female rat pituitary glands were dissociated, separated and enriched on a continuous gradient of bovine serum albumin at unit gravity. Two lactotroph subpopulations were observed in the light (F(3-5)) and the heavy (F(7-9)) fractions of the gradient. Both populations were maintained for at least 6 days in culture before experiments were performed. 2. Patch-clamp recordings, in the whole-cell mode, were performed on both lactotroph subpopulations in order to measure passive membrane properties and Ca2+ currents. Resting membrane potential as well as membrane capacitance values were found to be lower in light fraction cells. The two components of Ca2+ currents, called fast and slow deactivating (FD and SD) currents, were present with different proportions in each subpopulation; the ratio of current amplitudes, SD/FD, was 2.42 +/- 0.41 (n = 18) in light fraction cells and 1.17 +/- 0.27 (n = 17) in heavy fraction cells (P less than 0.02). 3. Reverse haemolytic plaque assay showed that in the light and heavy fractions, 68 and 47% of the lactotroph cells, respectively, were secreting. Population analysis of the plaque areas revealed a bimodal frequency distribution of plaque sizes consisting of small (1500 microns 2) and large plaques (3995 microns 2). A majority of light fraction cells produced large plaques whereas most of the heavy fraction cells produced small plaques. 4. Perifusion experiments performed on enriched prolactin cells showed that (1) basal prolactin (PRL) release was higher in light fraction than in heavy fraction cells, (2) the dopamine (10(-8)M)-induced inhibition of PRL release was greater in light fraction cells (86 +/- 15%) than in heavy fraction cells (41 +/- 21%), and (3) the thyrotrophin-releasing hormone (TRH, 10(-8)M)-induced increase of PRL release was 150 +/- 60% in light fraction versus 330 +/- 82% in heavy fraction cells. 5. Current-clamp recordings were performed using the intracellular technique. Lactotrophs were categorized according to their electrophysiological response following application of dopamine or TRH (both 10(-8)M). In the light fractions, the majority of the cells tested were hyperpolarized by dopamine (68%), whereas only 7% were depolarized by TRH application. In the heavy fractions, most of the cells (63%) responded to TRH application, while only 13% were dopamine sensitive. 6. Cytosolic free Ca2+ concentration ([Ca2+]i) measurements with the fluorescent probe Indo-1 revealed two lactotroph subtypes. Most cells in the light fractions (sixteen of twenty-two tested cells) exhibited an unstable level of [Ca2+]i with values fluctuating between 114.1 +/- 34.3 and 221 +/- 50 nM (mean +/- S.D.). Application of dopamine or of the D2 receptor agonist RU 24213 (10(-8)M) resulted in the disappearance of these fluctuations and in an accompanying decrease in basal [Ca2+]i level.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Voltage-dependent calcium and potassium channels in Schwann cells cultured from dorsal root ganglia of the mouse.

1. Whole-cell patch clamp studies were carried out on Schwann cells in organotypic cultures of dorsal root ganglia (DRG) from OF1 mice embryos (18-19 days). 2. In standard external solution, from a holding potential of -70 mV, two types of voltage-dependent K+ currents were recorded: a fast transient current and a delayed sustained current. With a holding potential of -30 mV, only the delayed sustained current could be evoked. 3. Both K+ currents were inhibited by tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP) in a dose-dependent manner. For the transient current the half-maximal effective dose was 100 mM for TEA and 1.3 mM for 4-AP. For the delayed sustained current the half-maximal effective dose was 11 mM for TEA and 4 mM for 4-AP. Both currents were insensitive to external Ca2+. 4. The delayed sustained current, isolated by use of a holding potential of -30 mV displayed a 'cumulative inactivation' which was removed by hyperpolarizing the membrane to -70 mV between each test pulse. 5. In K(+)-free external and pipette solutions, with 10 mM-external Ca2+, from a holding potential of -70 mV voltage-dependent Ca2+ channel currents were recorded. The threshold for activation was -45.3 +/- 5.4 mV (mean +/- S.D., n = 5) and the current inactivated fully at the end of the test potential. The current was unaffected by 2 microM-tetrodotoxin (TTX) and totally blocked by 5 mM-Co2+. 6. Equimolar replacement of external Ca2+ by Ba2+ did not significantly modify the voltage dependence (threshold for activation -42.8 +/- 6.4 mV, n = 7) or the magnitude of the inward current. Ca2+ and Ba2+ were equally permeant. The fully inactivating current was insensitive to both nifedipine and Bay K 8644 (1 microM each). Increasing the external Ba2+ concentration from 10 to 89 mM enhanced the Ba2+ current and shifted the voltage dependence of the current (threshold for activation, -30.5 +/- 7.3 mV, n = 9) along the voltage axis as expected for altered external surface potential. 7. In 89 mM-external Ba2+ solution, some cells displayed an additional slowly decaying current which was totally blocked by nifedipine (1 microM). 8. Ca2+ channel currents were recorded only when DRG neurons were present in the culture, as excision of explants and subsequent axonal degeneration led to loss of detectable Ca2+ channel currents. This phenomenon was never observed for K+ currents. 9. We conclude that mouse Schwann cells in organotypic culture possess voltage-dependent K+ and Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1991

Blockade by lithium ions of potassium channels in rat anterior pituitary cells.

Extracellular Li+ has been known to facilitate the basal secretion of growth hormone from anterior pituitary cells and of catecholamine from chromaffin cells. In both cases, the intracellular accumulation of Li+ seems to be the prerequisite, and the presence of extracellular Ca2+ is indispensable. In this series of experiments, we examined whether Li+ blocked K+ currents by using primary cultured anterior pituitary cells from male rats. K+ currents were measured in the whole cell configuration of the patch-clamp technique. Extracellular Li+ (140 mM) suppressed both the delayed rectifier K+ current (IK) and the transient outward K+ current to 71 and 69% of control, respectively, in a reversible manner. IK elicited by a voltage step to +70 mV from holding potential of -70 mV was suppressed by 32.5 mM internal Li+ to 28% of control. Half-maximal suppression of K+ conductance by internal Li+ was 16 mM. Furthermore, Ca(2+)-channel blocker methoxyverapamil potently suppressed Li(+)-induced growth hormone secretion. From these results we propose that the blockade by Li+ of K+ channels could depolarize the cells and activate Ca2+ channels, thereby promoting the influx of Ca2+ and hormone secretion as a mechanism of Li(+)-induced hormone secretion.

Animals↗
PubMed1991

Different expression of the two dopaminergic D2 receptors, D2415 and D2444, in two types of lactotroph each characterised by their response to dopamine, and modification of expression by sex steroids.

Dopamine inhibits prolactin liberation acting via the D2 type receptor. Two different electrophysiological responses to dopamine have been shown to characterise two types of lactotroph isolated from the lactating female rat. It is now known that differential splicing of the pre-messenger RNA coding for the D2 receptor leads to the production of two D2 subtypes, D2(415) and D2(444). These subtypes differ in the region which is believed to be responsible for the binding of G proteins, and could thus lead to the activation of different intracellular second messenger systems. Here we show that the pre-messenger RNA for the D2 receptor is differentially spliced in such a way that the ratio D2(415)/D2(444) is significantly different (2.91 +/- 0.6 vs 1.29 +/- 0.14) between two populations of lactotrophs, each enriched in cells showing one type of response to DA. We further show that the ratio D2(415)/D2(444) can be changed by treatment of prolactin cells in primary culture with progesterone or testosterone. Estrogen did not change the ratio, but diminished the total amount of D2 cDNA. Regulation of differential splicing by sex steroids could provide a mechanism for modifying lactotroph responsiveness to DA in different physiological situations.

Animals↗
PubMed1990

Evidence for a relationship between lactotroph heterogeneity and physiological context.

Two lactotroph subpopulations differing in their functional properties have previously been isolated from lactating female rats. It was found that adult female rats at proestrus similarly yield two subpopulations of lactotrophs, whereas those from adult female rats at metestrus show different properties and in males only one functional population is found. As the period of lactation and proestrus are characterised by high circulating estrogen levels, estrogen is considered to modify lactotroph properties in the female, and as the functional differences concern the lactotroph responsiveness to TRH, it is supposed that lactotroph heterogeneity provides a means for particular secretory patterns during lactation and at proestrus.

Animals↗
PubMed1990

A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium currents by dopamine in rat lactotrophs.

The present study examines the effect of dopamine (DA), known to inhibit prolactin (PRL) release, on voltage-activated calcium currents in identified rat lactotrophs. Two types of voltage-dependent Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. Both were reversibly inhibited by DA application. The inhibitory action of DA was reduced by (i) sulpiride (D2 antagonist), (ii) preincubation of the cells with pertussis toxin (PTX), and (iii) inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) in the pipette solution, whereas it was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). This DA-induced response could not be overcome by changing the adenosine 3',5'-cyclic monophosphate level. These findings suggest that DA can inhibit Ca2+ entry through voltage-activated Ca2+ channels via a PTX-sensitive G protein(s) pathway thereby affecting PRL release from rat lactotrophs.

Animals↗
PubMed1990

Sodium and calcium currents in action potentials of rat somatotrophs: their possible functions in growth hormone secretion.

We report that both Na+ and Ca2+ currents are involved in the action potentials and in the hormone release from rat somatotrophs in primary culture. Single somatotrophs were identified by reverse hemolytic plaque assay (RHPA) and transmembrane voltage and currents were recorded using the whole-cell mode of the patch-clamp technique. Somatotrophs displayed a mean resting potential of -80mV and an average input resistance of 5.7G omega. Most of the cells showed spontaneous or evoked action potentials. Single action potentials or the initial spike in a burst were characterized by their high amplitude and short duration. Tetrodotoxin (TTX, 1 microM) blocked single action potentials and the initial spikes in a burst, whereas action potentials of long duration and low amplitude persisted. Cobalt (2 mM) plus TTX (1 microM) blocked all the action potentials. Voltage-clamp experiments confirmed the presence of both a TTX-sensitive Na+ current and Co2(+)-sensitive Ca2+ currents. TTX or Na(+)-free medium slightly decreased the basal release of GH but did not markedly modify hGRF-stimulated GH release. However, Co2+ (2 mM), which partially decreased the basal release, totally blocked hGRF-stimulated release. We conclude that (1) Na+ currents which initiate rapid action potentials may participate in spontaneous GH release; (2) Ca2+ currents, which give rise to long duration action potentials and membrane voltage fluctuation, are probably involved in both basal and hGRF-stimulated GH releases.

Action Potentials↗
PubMed1990

Contribution of arachidonate metabolites to basal and thyrotropin releasing-hormone-stimulated release of prolactin from purified lactotrophs in primary culture.

Among the different biochemical pathways which have been suggested to play a role in the control of prolactin (Prl) release from anterior pituitaries, arachidonate and its metabolites have been proposed to be involved in the process of Prl release. In this study we investigated the contribution of arachidonate metabolites to both basal and TRH-stimulated Prl release from perifused lactotrophs in culture (derived from pituitary glands of lactating female rats), which exhibit a high sustained release of Prl in absence of inhibitory input. Inhibition of the general oxidative metabolism of arachidonate by 10(-5) M ETYA or of the arachidonate lipoxygenase metabolism by 10(-5) M NDGA decreased basal Prl release to 45 +/- 10% (n = 3) and 36 +/- 4% (n = 6) of the control release, respectively. Indomethacin, an inhibitor of the cyclooxygenase pathway, was without effect. Of the lipoxygenase metabolites tested at 10(-6) M only 15-HPETE and 15-HETE induced Prl release. 15-HETE elicited prolactin release in a concentration dependent manner with a maximal effect at 10(-6) M (10.72 +/- 3 ng/ml vs control 5.1 +/- 0.8 ng/ml, n = 3). The quantity of Prl release induced by TRH was markedly decreased in the presence of NDGA. However, the fraction of Prl release elicited by TRH, calculated as a percentage of the amount of Prl released prior to TRH application, was similar under control conditions, and in the presence of NDGA. In contrast, inhibition of the protein kinases A and G by H8 (10(-5) M) failed to alter basal Prl release but inhibited the effect of TRH by 58 +/- 6% (n = 3). These data suggest that in absence of inhibitory inputs the high sustained release of Prl observed in cultures of lactotrophs derived from lactating female rats depends on the availability of lipoxygenase metabolites, and that the blockade of lipoxygenase reduces the absolute amount of Prl released by TRH without suppressing the ability of TRH to stimulate Prl release.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

Two types of voltage-dependent calcium current in rat somatotrophs are reduced by somatostatin.

1. Somatotrophs were obtained from rat pituitary glands after dissociation, separation and enrichment on a continuous gradient of bovine serum albumin at unit gravity. Somatotrophs were enriched up to 85% in the heavy fractions (F8 and F9). 2. After identification by reverse hemolytic plaque assay, patch-clamp recording in the whole-cell mode was performed on somatotrophs. 3. Under voltage-clamp conditions, two types of Ca2+ currents were recorded. From a holding potential of -70 mV, depolarizing voltage steps to potentials more positive than -50 mV activated a current which rapidly inactivated and which was very sensitive to Ni2+ but not to Cd2+. This current corresponds to T-type current. Depolarizing steps to potentials more positive than -30 mV from a holding potential of -40 mV triggered a current which slowly inactivated and which was very sensitive to Cd2+ but not to Ni2+. This current corresponds to L-type current. 4. Application of somatostatin to the bath solution (10 nM) markedly reduced the amplitudes of both T- and L-type currents. Somatostatin decreased the conductance of L-type current without modifying its time- and voltage-dependent inactivation but its activation was not affected. However, somatostatin decreased the conductance of T-type currents, and also accelerated its time-dependent inactivation. Half-inactivation voltage of T-type current was shifted from -52 to -63 mV by somatostatin but no change was obtained in the current activation curve. 5. All these modifications in Ca2+ currents were abolished by a pre-treatment of the cultures with pertussis toxin (100 ng/ml, for 10 h). This pre-treatment also blocked the inhibitory effect of somatostatin on high-K(+)-stimulated growth hormone release. 6. Our results show that somatostatin acts on somatotrophs by attenuating the voltage-dependent Ca2+ currents. These effects may contribute to a somatostatin-induced reduction in [Ca2+]i and the subsequent decline in growth hormone release.

Animals↗
PubMed1990

Somatostatin increases voltage-dependent potassium currents in rat somatotrophs.

To study the modulatory effects of somatostatin on membrane K+ currents, whole cell voltage-clamp recordings were performed on identified rat somatotrophs in primary culture. In the presence of Co2+ (2 mM) and tetrodotoxin (1 microM) in the bath solution to block Ca2+ and Na+ inward currents, two types of voltage-activated K+ currents were identified on the basis of their kinetics and pharmacology. First, a delayed rectifier K+ current (IK) had a threshold of -20 mV, did not decay during voltage steps lasting 300 ms, and was markedly attenuated by extracellular application of tetraethylammonium (TEA, 10 mM). Second, a transient outward K+ current (IA) was activated at -40 mV (from a holding potential of -80 mV) and persisted despite the presence of TEA. This IA was blocked by 4-aminopyridine (2 mM). Somatostatin (10 nM) increased IK by 75% and IA by 45% without obvious effects on steady-state voltage dependency of activation or inactivation, and these effects were reversible. This increase in K+ currents may contribute in part to the inhibitory effect of somatostatin on growth hormone release.

4-Aminopyridine↗
PubMed1990

Plateau potentials recorded from lactating rat enriched lactotroph cells are triggered by thyrotropin releasing hormone and shortened by dopamine.

Enrichment of dispersed pituitary cells from normal lactating rats on a continuous BSA gradient permitted the isolation of two prolactin cell populations--light and heavy. Hormone release studies indicated that spontaneous prolactin (PRL) release of the heavy fraction cells was particularly sensitive to TRH stimulation (mean of 450%) and that this effect of TRH was totally inhibited by Ca2+ channel blockers (Co2+, Cd2+, Ni2+ and Mn2+). For this reason, the electrophysiological response to TRH was investigated on heavy fraction cells. Experiments performed on 264 cells after 4-12 days in culture showed that these cells could be divided into two groups. The first group, called high resting potential (HRP) cells, constituted 73% of the total and was characterized by a mean resting potential of -60 mV and a mean input membrane resistance of 700 M omega, and these cells displayed plateau potentials, which were triggered by application of brief (2 s), large (1 nA) depolarizing or hyperpolarizing current steps. The plateaux were characterized by a sustained depolarization at a potential near -20 mV and they were concomitant with an increase of the membrane conductance. The repolarization consisted of a slight, gradual hyperpolarization followed by a rapid return to the resting potential. The second group of cells (the remaining 27%; called low resting potential or LRP cells) was characterized by a mean resting potential of -45 mV and a mean input membrane resistance of 250 M omega. These cells were excitable, 30% of them displaying spontaneous activity but never showing plateaux. Electrophysiological experiments showed that the majority of the HRP cells (99%) responded to TRH but were insensitive to dopamine (DA) ejections (up to 10(-6) M) at resting potential. Ejection of TRH onto HRP cells induced a slow depolarization (10-15 mV) concomitant with a decrease of the membrane resistance. This led the cell membrane potential to a critical value (approximately -50 mV), at which the plateau response was triggered. The plateau lasted for about 10 s from potentials greater than -100 mV, and could reach 10 min from holding potentials close to the resting potential. The amplitude of the plateau varied according to the holding potential and the reversal potential was found to be -20 mV. Local application of tetraethylammonium chloride (TEA, 30 mM) only slightly affected the amplitude of the plateaux but they were shortened or totally blocked by Co2+, Cd2+ and Ni2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗