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B Dufy

Publications and source records attributed to B Dufy.

At least 19 recordsLinked to original sources
PubMed1992

Calcium-activated chloride conductance of lactotrophs: comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation.

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.

Animals↗
PubMed1992

Continuous culture of neuronal cells from adult human olfactory epithelium.

Cells from the olfactory epithelium of adult human cadavers have been propagated in primary culture and subsequently cloned. These cells exhibit neuronal properties including: neuron-specific enolase, olfactory marker protein, neurofilaments, and growth-associated protein 43. Simultaneously, the cells exhibit nonneuronal properties such as glial fibrillary acidic protein and keratin, the latter suggesting properties of neuroblasts or stem cells. These clonal cultures contain 5-10% of cells sufficiently differentiated to show odorant-dependent cyclic adenosine 3',5'-monophosphate (cAMP) or calcium-release responses when challenged with submicromolar concentrations of odorants. The potential of culturing neuronal cells from patients with neuropsychiatric disorders, such as Alzheimer's disease or schizophrenia, could enable the study of the pathophysiology of these neurons in the culture dish and allow new approaches to the study of mental illness.

Adult↗
PubMed1992

Complex effects of arachidonic acid and its lipoxygenase products on cytosolic calcium in GH3 cells.

The mechanisms by which arachidonic acid (AA) and its metabolites stimulate prolactin release from pituitary cells are not understood. Because Ca2+ ions are pivotal to exocytosis, we investigated the effects of AA metabolites on intracellular calcium concentration ([Ca2+]i) and membrane ionic currents using dual-excitation microspectrofluorimetry and whole cell patch-clamp techniques in GH3/B6 pituitary line cells. AA (1 microM) had a biphasic effect on [Ca2+]i, mobilization of intracellular Ca2+, followed by stimulation of Ca2+ entry. Only the latter appeared to result from the degradation of AA through the lipoxygenase pathway. Indomethacin (Indo, 10 microM) and lipoxygenase products 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE, 1 microM), increased action potential duration (12-HETE) or frequency (5- and 15-HETE, Indo). These effects depended on inhibition of d-tubocurarine- or tetraethylammonium-sensitive K+ conductances and stimulation of voltage-dependent Ca2+ channels. Refilling of intracellular Ca2+ stores, and Ca2+ efflux, may also be stimulated. Our results demonstrate a control of [Ca2+]i by a second messenger (AA) and its metabolites (HETEs).

Arachidonic Acid↗
PubMed1992

Voltage-dependent calcium current in human decidual cells and its relation to prolactin secretion.

Human decidual cells synthesize and release decidual PRL (dPRL) immunologically and biochemically identical to human pituitary PRL. However, stimulators and inhibitors of PRL secretion such as TRH, bromocriptine or dopamine have no effect on dPRL release. The evidence for the involvement of Ca2+ in dPRL release is based on contradictory or unclear data. Since little is known about Ca2+ movement in human decidual cells we studied the membrane Ca2+ conductance of cultured decidual cells using the patch-clamp technique in the whole-cell configuration. We report the existence of Ca(2+)-dependent action potentials triggered by hyperpolarizing or depolarizing pulses and blocked by cobalt (Co2+; 5 mM). Spontaneous action potentials were observed in the cell-attached mode and found also to be Co(2+)-sensitive. A tetrodotoxin-insensitive and Ca(2+)-dependent rapidly inactivating inward current was investigated in voltage clamp. Its activation threshold was between -60 and -45 mV. Indo-1 measurements of free intracellular Ca2+ concentrations ([Ca2+]i, 169 +/- 14 nM and 141 +/- 8 nM in short-term culture vs. 149 +/- 5 nM in cells cultured for 3-6 days) showed that decidual cells have spontaneous transient fluctuations of [Ca2+]i and that [Ca2+]i was decreased by Ca2+ channel blockers. The existence of Ca2+ movements in decidual cells in culture is thus demonstrated. The occurrence of action potentials in decidual cells derived from fibroblasts, reputed to be inexitable cells, is an interesting biological observation. However, Ca2+ is not involved in the short-term release of PRL by decidual cells, and its effects on long-term secretion still requires further investigation.

Action Potentials↗
PubMed1992

Calcium homeostasis in growth hormone (GH)-secreting adenoma cells: effect of GH-releasing factor.

Human GH-secreting tumors are heterogenous regarding their basal secretory activity and response to GH-releasing factor (GRF). We have investigated whether such different secretory properties could be accounted for by alterations of intracellular mechanisms occurring at the calcium level. Basal free intracellular calcium concentrations ([Ca2+]i) and Ca2+ responses to GRF were studied in single cells cultured from fragments of five GH-secreting pituitary adenomas. We used the microspectrofluorimetric method and indo-1 as the fluorescent probe. The cell populations cultured from the tumors of patients A and C showed increased hormone secretion in response to GRF in vitro, whereas cultures from patients B, D, and E were unresponsive to the peptide. Basal [Ca2+]i measured in the five cell populations ranged from 82 +/- 18 to 118 +/- 27 nM. A 10-sec application of 10 nM GRF induced an increase in [Ca2+]i in 60% and 54% of A and C cells, respectively. In the nonresponsive cell populations, the number of calcium responses to GRF was lower, 26% (B cells), 5% (D cells), and 10% (E cells). Two principal responses types were observed: 1) an initial increase in [Ca2+]i, followed by a sustained plateau phase lasting for more than 200 sec; and 2) a monophasic peak of increased [Ca2+]i lasting approximately 1 min before returning to baseline levels. GRF responses were totally suppressed in the absence of Ca2+ ions in the external medium. Sixteen to 30% of the cells cultured from four of the five tumors showed spontaneous fluctuations of [Ca2+]i. These spontaneous Ca2+ transients were suppressed in Ca(2+)-free medium. The number of cells exhibiting such Ca2+ transients decreased with time in culture. Basal hormone secretion was higher in cultures from patient D, in which no spontaneous Ca2+ transients were observed in any of the 72 studied cells, and in cultures from patients E, in which only 16% of cells were spontaneously active. We conclude that 1) in human responsive somatotrophs, the involvement of Ca2+ in GRF stimulus-secretion coupling mechanisms is apparently similar to that described in somatotrophs of other species; 2) the lack of a secretory response to GRF observed in some tumors may result from impairment of Ca2+ responsiveness in either cell recruitment or response amplitude and/or duration; and 3) spontaneous rhythmic Ca2+ activity is apparently dissociated from basal hormone secretion in some of these tumor cells.

Adenoma↗
PubMed1992

Multihormonal response to corticotropin-releasing hormone in inferior petrosal sinus blood of one patient with Cushing's disease: comparison with in vitro secretion of the tumoral corticotropes.

A multihormonal response to CRH during inferior petrosal sinus sampling in patients with Cushing's disease has recently been described. Whether it reflects multihormonal secretion by the corticotropic adenoma, or secretion by non-tumorous adjacent cells via paracrine mechanisms remains debatable. We have compared the effect of CRH on ACTH, GH, PRL and TSH secretion during inferior petrosal sinus sampling with its effect on the in vitro secretion of the corticotropic adenoma after excision in one case of Cushing's disease. Before CRH injection in vivo results show significant central-peripheral gradients for all hormones but only ACTH lateralized to the side of the tumor. After CRH administration, the petrosal concentrations of all hormones increased preferentially on the side of the adenoma resulting in significant intersinus gradients: 8.1 for ACTH, 2.0 for GH, 1.8 for PRL and 1.5 for TSH. In vitro results: the adenoma cells were immunostainable for ACTH only. In culture, they secreted ACTH only. Addition of CRH to the culture induced a mean increase of 160% in ACTH secretion but GH, PRL and TSH remained undetectable. Our results favor the hypothesis that the multihormonal response to CRH seen during inferior petrosal sinus sampling in Cushing's disease reflects a paracrine stimulation of the adjacent non-tumorous pituitary cells by the corticotropic adenoma.

Adenoma↗
PubMed1991

Intracellular pH in individual pituitary cells: measurement with a dual emission pH indicator.

Intracellular pH (pHi) can now be measured at the single cell level using dual emission wavelength microspectrofluorimetry with the fluorescent pH indicator SNARF 1 and its membrane permeant acetoxymethyl ester (SNARF 1/AM). We measured pHi of individual pituitary cells under both basal and stimulated conditions. The emitted fluorescence of SNARF 1 probe was calibrated following experimental manipulations of pHi in two types of rat pituitary cells. The calibration curves obtained in the two cell types were identical. We observed a Gaussian distribution of individual pHi with a wide dispersion (6.95 to 8) in the two cell populations. TRH (10(-7) M) and ionomycin (5 microM) induced a transient acidification followed by a sustained alkalinization, whereas K+ (50 mM) depolarization only exerted a transient acidification. These results show that the dual emission pH indicator SNARF 1 can be used to reliably investigate changes in pHi in individual endocrine cells.

Animals↗
PubMed1991

Microencapsulation of isolated pituitary cells by polyacrylamide microlatex coagulation on agarose beads.

Microlatex beads of homogenous size were made by polymerization of a mixture of acrylamide/bisacrylamide dispersed in a microemulsion. The microlatex was aggregated by dilution of the microemulsion in acrylamide solutions. The aggregates were then coagulated by polymerization at the interfaces of agarose beads circulating in a capillary tube containing paraffin oil. Biocompatibility was tested on isolated pituitary cells microencapsulated by this procedure.

Acrylamide↗
PubMed1991

The gonadotropin-releasing hormone associated peptide reduces calcium entry in prolactin-secreting cells.

The precursor molecule to the GnRH contains a peptide named GnRH-associated peptide (GAP) with PRL-inhibiting properties. In this work, we have studied the electrophysiological properties and responses to GAP of three different types of PRL-secreting cells: 1) the rat tumor cell line GH3, 2) normal rat pituitary cells in primary culture, and 3) human PRL-secreting adenoma cells. Using different but complementary techniques we show that GAP reduces intracellular Ca++ levels, [Ca++]i, and inhibits Ca++ transients in these cells. This reduction of [Ca++]i results from coordinate actions of GAP on K+ and Ca++ conductances and may explain the inhibitory effect of GAP on hormonal secretion by PRL-secreting cells.

Adenoma↗
PubMed1991

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line.

The GH3 pituitary cell line has been extensively used to study various aspects of the stimulus secretion coupling process. It is known that GH3 cells release PRL and GH in the basal state and in response to various secretagogues. However, this cell line was considered unsuitable as a model for studying the effects of GHRF since the neuropeptide did not affect GH secretion or gene expression. This suggested that the GH3 cells may lack GHRF receptors. The present study investigates the effect of GHRF on free intracellular Ca2+ concentrations in GH3 cells. Cytosolic free calcium concentrations ([Ca2+]i) were monitored in individual cells by microspectrofluorimetry using the fluorescent dye indo 1. When the cells were challenged with a brief application of GHRF (100 nM; 15 sec), 36 out of 59 of these cells responded within a few seconds by a marked increase in [Ca2+]i. GHRF enhanced the frequency of [Ca2+]i oscillations in spontaneously active cells or triggered [Ca2+]i oscillations in inactive cells. The response to GHRF was totally blocked by external Ca2+ free solutions and Ca2+ channel blockers. Combined electrophysiological and fluorescent experiments were carried out in 16 cells. Eleven responded to GHRF. In all cases, the Ca2+ transients triggered by GHRF were associated with action potentials. The Ca2+ responses observed in our experiments clearly show that GH3 cells possess membrane receptors to GHRF. Thus, it is likely that the lack of secretory response observed in GH3 cells does not result from the absence of binding sites to the peptide. It is more likely to be related to alterations of transduction mechanisms resulting in uncoupling between stimulation and secretion.

Calcium↗
PubMed1991

[Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells].

We have investigated the electrical response of patched GH3 cells to Growth-Hormone Releasing-Hormone (GH-RH). GH-RH (100 nM) enhanced firing frequency of action potentials. This is accompanied by membrane depolarization (5-10 mV) and conductance increase. Voltage clamp studies reveal that GH-RH potentiates calcium inward currents and a calcium-dependent chloride current; transient outward current is diminished. These changes in membrane conductance account for the cytosolic free calcium rise shown by Indo-1 fluorescence measurements.

Action Potentials↗
PubMed1990

Thyrotropin-releasing hormone activates a [Ca2+]i-dependent K+ current in GH3 pituitary cells via Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive mechanisms.

The role of Ins(1,4,5)P3 in receptor-induced Ca2+ mobilization in pituitary cells was studied at the single-cell level. Experimental strategies were developed which allowed a comparative analysis of the effects of Ins(1,4,5)P3 with those of receptor activation under identical conditions. These include microfluorimetry as well as a novel technique which permits the controlled and rapid application of intracellular messenger molecules to individual cells. This latter approach is based on the tight-seal whole-cell recording (WCR) technique, and utilizes two patch-clamp micropipettes, one for electrical recording and the second for the controlled pressure injection. Ins(1,4,5)P3, when applied with this dual-WCR (DWCR) technique, leads rapidly to a marked rise in cytosolic free Ca2+ [( Ca2+]i) and a concomitant stimulation of Ca2(+)-activated K+ current; Ins(1,4,5)P3 can thus mimic the effects of thyrotropin-releasing hormone (TRH) in the same cells under identical conditions. In cells dialysed intracellularly with heparin, a potent antagonist of Ins(1,4,5)P3 action, the rapid response to extracellular stimulation with TRH was abolished, as were the effects of intracellular application of Ins(1,4,5)P3. Heparin, which abolished Ins(1,4,5)P3 action completely, blocked responses to TRH in some cells only partially, revealing that Ca2+ mobilization response to TRH is in part slower in onset than the response to Ins(1,4,5)P3. It is concluded (1) that Ins(1,4,5)P3 is an essential element for the action of TRH, providing a rapid mechanism for Ca2+ mobilization induced by the releasing hormone and (2) that TRH action in mobilizing intracellular Ca2+ is sustained by a slower mechanism which is independent of Ins(1,4,5)P3.

Animals↗
PubMed1990

Electrophysiological response to thyrotropin-releasing hormone of rat lactotrophs in primary culture.

The response of rat pituitary cells to thyrotropin-releasing hormone (TRH) in primary culture was studied in the whole-cell configuration with the patch-clamp technique. Prolactin (PRL)-containing cells were identified in the culture with a peroxidase-antiperoxidase immunocytochemical method. The cells were cultured from the pituitaries of diestrous (D) and lactating (L) female rats. Membrane electrophysiological properties (resting potential and input resistance) of pituitary cells in primary culture varied widely. Under the recording conditions reported here, the mean resting potential of lactotrophs was about -30 mV. There were spontaneous fluctuations in membrane resting potential (10-15 mV) as well as of membrane input resistance, making these parameters difficult to evaluate accurately. Most of the cells exhibited spontaneous firing activity that was shown to be mainly calcium-dependent. There was no difference between L and D cells in resting membrane electrophysiological properties. TRH (10(-7) M) induced a transient hyperpolarization of the membrane similar to that previously described in the GH3 clonal pituitary cell line. Voltage-clamp studies showed that this hyperpolarization resulted from activation of an outward current, the reversal potential of which ranged from -48 to -86.5 mV. Experimental manipulations of the ionic composition of internal and external recording media suggested that both K+ and Cl- were involved. This hyperpolarizing response was observed both in D and L cells, although L cells had larger and faster responses than D cells. This observation may be of physiological significance because lactotrophs have been reported to exist in various subtypes.

Action Potentials↗
PubMed1989

Measurement of CA2+ transients using simultaneous dual-emission microspectrofluorimetry and electrophysiology in individual pituitary cells.

Cytosolic free calcium concentration, [Ca2+]i, was monitored in individual pituitary GH3B6 cells, loaded with the fluorescent Ca2+ indicator indo 1 either by internal perfusion through a patch clamp pipette or by exposure to indo 1 acetoxymethyl ester, with the use of a dual-emission apparatus for microspectrofluorimetry. This system was sensitive enough to allow on-line monitoring of [Ca2+]i (from the ratio of fluorescent intensities) which could be combined simultaneously with whole-cell patch clamp recordings. The following situations were examined: (a) [Ca2+]i oscillations due to action potential firing, and (b) rapid transient elevations of [Ca2+]i triggered by voltage-dependent Ca2+ current. The results indicate that monitoring of [Ca2+]i at the single cell level with indo 1 provides a powerful means to study the [Ca2+]i regulation in pituitary cells, which should be applicable to many other cell types.

Action Potentials↗
PubMed1989

Inhibitors of 1,2-diacylglycerol kinase potentiate the TRH-induced stimulation of Ca2+-activated K+ current.

Transient activation of the outward K+ current caused by a rise in the cytosolic free Ca2+ concentration, [Ca2+]i was the predominant change in plasma membrane ion flux during the first phase of thyrotropin-releasing hormone (TRH) action on pituitary cells. Following the intracellular application of inhibitors of 1,2-diacylglycerol (DG) kinase, R59022 and 1-oleyl-2-acetyl glycerol (OAG) the outward K+ current response to TRH in cells of the pituitary line GH3B6 was potentiated. This potentiation was analyzed further with the combination of microfluorimetric and electrophysiological recording techniques. Receptor-induced changes in [Ca2+]i and ion channel activation were monitored simultaneously in the same cell. It was found that R59022 and OAG altered in parallel the TRH-induced transient rise in [Ca2+]i and outward K+ current. This resulted in a significant correlation between the kinetic parameters (speed of onset, duration) of the [Ca2+]i and the K+ current responses to TRH. Intracellular application of vanadate abolished the rapid start of the TRH response presumably by its block of Ca2+ uptake into the endoplasmic reticulum, leading to depletion of a Ca2+ pool mobilizable by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The use of vanadate unmasked a slowly developing response to TRH, which was still potentiated by OAG and R59022. Together, these observations suggest that Ca2+ mobilization during the first phase of TRH action is mediated by two distinct processes, one of which is linked to receptor stimulation of DG production.

Calcium↗
PubMed1989

Arachidonic acid affects membrane ionic conductances of GH3 pituitary cells.

Arachidonic acid (AA) stimulates prolactin release from pituitary cells, by mechanisms not yet understood. In this work, we analyzed the effects of AA on membrane ionic conductances in a clonal line of anterior pituitary cells (GH3/B6), finding time- and dose-dependent effects of AA on their membrane ionic conductances. The predominant response at concentrations between 100 nM and 10 microM was a prolongation of the action potential (AP) and an increase in the transient after-hyperpolarization potential. Voltage clamp studies showed that this was associated with a decrease in a voltage-dependent potassium current and an increase in a voltage-dependent calcium current. In some cells (30%) the effect of AP duration was less important, but spike firing was enhanced. For the highest concentrations used (1 and 10 microM) the effects described above were preceded by hyperpolarization of the cell membrane; in voltage clamp it was shown that this hyperpolarization resulted from the activation of a calcium-dependent potassium conductance suspected to be due to the release of intracellular calcium. The calcium store affected by AA was, at least in part, insensitive to vanadate and heparin. These data suggest that AA may enhance intracellular calcium concentration by increasing calcium entry during each voltage-dependent calcium AP, by increasing the spike frequency, or by releasing calcium from an intracellular compartment. The resulting rise in cytosolic free calcium concentration may be a key link in the process by which AA stimulates prolactin release in GH3/B6 pituitary cells.

Animals↗