Biomedical subjects

B Dufy

Publications and source records attributed to B Dufy.

At least 37 records · Page 2Linked to original sources

PubMed1995

Recording of a large-conductance chloride channel in normal rat lactotrophs.

Membrane current fluctuations resembling channel openings and closings were observed in the whole cell configuration of the patch-clamp technique in normal rat lactotrophs in primary culture. Using high-gain head stage in whole cell configuration, we characterized the nature and pharmacological properties of the ionic channel underlying these fluctuations. This channel, found in small numbers (< 10 per cell), was specific for Cl- because its reversal potential varied with Cl- gradients, according to the Nernst equation, and its unitary amplitude was linearly related to membrane potential from -100 to 0 mV. Slope conductance was close to 100 pS. Analyzing open times, we demonstrated its Ca2+ and potential dependence. Four sublevels were observed. We suggest that this channel, belonging to the background Cl- channel group, takes part in the regulation of intracellular Cl- concentration of normal rat lactotrophs.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Voltage-dependent Ca2+ channels in Chinese hamster ovary (CHO) cells.

Voltage-dependent Ca2+ channels were identified in CHO-K1 cells, currently used in molecular biology studies. Experimental data obtained at both macroscopic and single-channel levels using the patch-clamp technique show that the Ca2+ current in CHO cells is similar to the high-threshold L-type of Ca2+ current previously observed in excitable cells. It can be carried by Ca2+ or Ba2+ ions, blocked by both inorganic (Co2+) and organic (nifedipine, isradipine) Ca2+ channel blockers. The unitary Ca2+ channel activity was characterized by a conductance of 19 pS in 60 mM Ca2+, by single exponential distribution of open times (t = 0.84 ms) and biexponential distribution of closed times (tf = 1.67 ms, ts = 7.9 ms). However, the functional role of these Ca2+ channels in CHO cells remains unclear.

Animals↗

PubMed1994

Voltage-dependent ionic conductances in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are becoming a widely used biological material. A number of studies report membrane ion conductance changes after transfection of channels and receptors, but there are few data available on the properties of membrane ion conductances of CHO cells before transfection. In this work we studied voltage-dependent ionic conductances in cultures of CHO native (CHO-K1) cells. Three types of voltage-dependent ionic conductances were identified: 1) a K+ conductance showing sensitivity to Ca2+ and a unit conductance of approximately 210 pS in symmetrical 150 mM K+ outside-out patches (this conductance, which did not inactivate during a 160-ms pulse, was inhibited by 30 nM charybdotoxin but not by 30 mM extracellular tetraethylammonium); 2) a rapidly activating and inactivating tetrodotoxin (TTX)-sensitive inward current, peaking at about -10 to 0 mV (this current showed characteristics similar in many respects to Na+ current recorded in neurons); and 3) another voltage-dependent inward current, which had slow inactivation, was TTX insensitive but was blocked by Co2+ (current was also carried by Ba2+, peaked at approximately 0 to +10 mV, was identified as a Ca2+ conductance, and was inhibited by dihydropyridines but not by 10 microM omega-conotoxin). Cell-attached patch recordings of single Ca2+ channel currents demonstrated a unitary conductance of approximately 20 pS.

Animals↗

PubMed1994

Early effects of PRL on ion conductances in CHO cells expressing PRL receptor.

Chinese hamster ovary (CHO-K1) cells were stably transfected with prolactin (PRL) receptor cDNA. These cells (CHO-E32) expressed the long form of functional PRL receptor. Using microfluorimetric and patch-clamp techniques, we have investigated the effects of PRL on intracellular Ca2+ concentration ([Ca2+]i) and membrane ion conductances. Exposure of CHO-E32 cells to 5 nM PRL resulted in an increase in [Ca2+]i. Two types of response were observed: 1) a stimulation of Ca2+ entry and 2) an intracellular Ca2+ mobilization. As PRL inhibited voltage-activated Ca2+ current, the PRL-induced Ca2+ increase does not involve voltage-activated Ca2+ channels. PRL also increased a charybdotoxin-sensitive Ca(2+)-dependent K+ conductance. Simultaneous measurements showed that PRL hyperpolarized the membrane potential before increasing intracellular Ca2+ levels. In voltage clamp, hyperpolarizing voltage steps were associated with increased Ca2+ concentrations, whereas depolarizing voltage steps decreased [Ca2+]i. Cell-free patch-clamp experiments showed that PRL directly stimulates K+ channel activity. Our results suggest the existence of a regulatory complex involving a protein kinase tightly associated with the Ca(2+)-activated K+ channels and that PRL stimulates these channels by means of the activation of protein kinase. The resulting hyperpolarization stimulates Ca2+ entry, probably through voltage-insensitive nonspecific channels.

Animals↗

PubMed1994

Short applications of gamma-aminobutyric acid increase intracellular calcium concentrations in single identified rat lactotrophs.

We have investigated the direct effect of GABA receptor agonists on the cytosolic free calcium concentration ([Ca2+]i) and the membrane potential of rat lactotrophs in primary culture. [Ca2+]i was recorded in single identified lactotrophs by dual emission microspectrofluorimetry using indo-1 as intracellular fluorescent calcium probe. Whole cell and perforated patch-clamp were performed. A short application of GABA (10(-5) M, 10 s) induced a marked transient [Ca2+]i increase in 66% of lactotrophs, which could be readily mimicked by muscimol (10(-5) M). By contrast, neither L-homocarnosine (10(-3) M) nor baclofen (10(-5) M), a GABAB agonist, had any effect on [Ca2+]i. The GABA-induced [Ca2+]i increase was antagonized by picrotoxin (10(-5) M), bicuculline methiodide (10(-5) M) and strychnine (10(-4) M), demonstrating GABAA receptor specificity. Furthermore, clonazepan (1.5 x 10(-4) M) could potentiate the GABA effect on [Ca2+]i. The [Ca2+]i increase disappeared in the absence of Ca2+ in the extracellular medium or in the presence of Ca2+ channel blockers (cadmium, PN 200-110). GABA and muscimol depolarized the membrane potential with a concomitant fall in cell input resistance, thus suggesting, as in other cell types, the opening of receptor-operated chloride channels. When Ca2+ entry was prevented by the use of cadmium (500 x 10(-6) M), GABA still elicited membrane depolarization but did not raise [Ca2+]i. Our results suggest that a short application of GABA leads to Ca2+ entry through voltage-gated Ca2+ channels in single lactotrophs. This Ca2+ influx is due to depolarization of the prolactin cell.

Animals↗

PubMed1994

Short term effect of prolactin on intracellular calcium in Chinese hamster ovary cells stably transfected with prolactin receptor complementary deoxyribonucleic acid.

The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined the effects of PRL on the intracellular free Ca2+ concentration in Chinese hamster ovary cells overexpressing functional PRL receptors. [Ca2+]i was determined by dual emission microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. We demonstrate that at physiological concentrations (0.5-5 nM), PRL stimulates Ca2+ entry (type I) and/or induces a mobilization of calcium ions stored in intracellular compartments (type II). Two types of Ca2+ mobilization, distinguishable by their onset kinetics, were observed, a slow mobilization (type IIa; transition time to peak, approximately 10 sec) and a fast mobilization (type IIb; transition time to peak, < 2 sec). PRL responses were delayed (15-120 sec) compared to the well known activation by phosphatidylinositol 4,5 bisphosphate hydrolysis-coupled receptors. This suggests that inositol trisphosphate is not involved in PRL response or that phosphatidylinositol 4,5 bisphosphate hydrolysis is not directly coupled to the PRL receptor. The amplitude of the PRL-induced Ca2+ increases (300-1400 nM) would be sufficient to provoke several physiological responses, such as stimulation of secretion, cell proliferation, or gene activation. However, the relation between the increase in Ca2+ and activation of milk protein genes remains to be established.

Animals↗

PubMed1994

Prolactin receptor and signal transduction to milk protein genes.

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 microM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the beta lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

Animals↗

PubMed1993

GnRH-associated peptide decreases cyclic AMP accumulation in the GH3 pituitary cell line.

We have studied the effects of GnRH-associated peptide (GAP) on cAMP, cGMP production and intracellular calcium (Ca2+) levels in the clonal rat GH3 pituitary cell line. We show that GAP decreases cAMP production in a dose-dependent manner. A 20-min incubation with GAP at concentrations greater than 0.1 nM resulted in a significant decrease in cAMP production. 100 nM GAP decreased cAMP production to 65% of that in control. The kinetics of the GAP effect were also studied. We show that cAMP levels fall to 68% of control levels within 2 min following administration of 10 nM GAP. Maximal inhibition was obtained after 3 min. The effect was only transient since GAP had no significant residual effect after 40 min. Conversely, up to 100 nM GAP failed to affect cGMP levels. We have also studied the effect of GAP on [Ca2+]i levels and showed that GAP decreases [Ca2+]i by inhibiting Ca2+ transients. GAP also reduced VIP-stimulated cAMP production and inhibited Ca2+ transients induced by VIP. The effect of GAP on cAMP production and Ca2+ transients was abolished by pertussis toxin treatment. These observations are consistent with GAP inhibiting PRL secretion through a cAMP, Ca(2+)-dependent process.

Animals↗

PubMed1993

Biphasic changes in intracellular pH induced by thyrotropin-releasing hormone in pituitary cells.

We studied the effects of TRH on intracellular pH (pHi) in individual cells of the GH3 pituitary clonal cell line using the seminaphtorhodafluor pH indicator. We show that, in a majority of cells, TRH action on pHi occurs in two phases: first acidification then alkalinization. Acidification and Ca2+ mobilization are related in time. K+ depolarization (KCl, 50 mM), and Ca2+ ionophores, A23187 (10 microM) or ionomycin (5 microM) lead to acidification. We conclude that a marked increase in [Ca2+]i can induce acidification and that the TRH-induced acidification is due to Ca2+ mobilization. TRH-induced alkalinization is due to Na+/H+ exchanger activation, since it is inhibited by amiloride (200 microM) and Na(+)-free medium. We show that this alkalinization does not occur after a 20-h pretreatment with phorbol myristate acetate (1 microM) which depletes protein kinase C. We also show that blocking Ca2+ entry does not affect the TRH-induced alkalinization, but an increase in [Ca2+]i concomitant with the activation of protein kinase C mimics TRH-induced alkalinization. We conclude that both Ca2+ mobilization and protein kinase C activation are necessary for TRH-induced alkalinization. Studies of secretion in Na(+)-free medium or with amiloride (200 microM) show that pHi does not seem to be involved in PRL short-term release (30 min) but suggest that activation of the Na+/H+ exchanger leading to cytoplasmic alkalinization may have an important role in PRL synthesis.

Amiloride↗

PubMed1993

Gonadotropin-releasing hormone-induced changes of intracellular pH in pituitary gonadotrophs: influence of estradiol.

Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.

Amiloride↗

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↗