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B Rossignol

Publications and source records attributed to B Rossignol.

At least 37 records · Page 2Linked to original sources
PubMed1996

Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: cellular distribution of muscarinic receptors.

The secretion of proteins and fluids from the exorbital lacrymal gland of rat is mainly controlled by muscarinic receptors. In a recent pharmacological study. Mauduit et al (Am J Physiol (1993) 264, C1550-C1560) identified a homogeneous population of M3 muscarinic receptors in preparations of acini from these tissues. In order to define the cellular composition of these acini and localize the muscarinic receptors, we have performed an immunofluorescent labelling study combined with confocal scanning microscopy. Antibodies raised against components of the different cytoskeletal networks (alpha-smooth muscle actin, cytokeratin peptide 14 and alpha-tubulin) revealed the presence of two different cell types. Cells with a stellate form are identified as myoepithelial cells, whereas rounded cells are secretory acinar cells. Both cell types are reactive with an antibody specifically directed against the muscarinic receptor. However, myoepithelial cells appear more intensely labelled than acinar cells. The roles of myoepithelial cells and secretory cells in the physiological function of the gland are discussed in terms of the distribution of muscarinic receptors.

Actins↗
PubMed1996

[Calcium transport in microsomes isolated from rat parotid gland. Effects of ADP and an ATP-regenerating system].

Calcium loading of a rat parotid microsomal fraction is greatly increased by an ATP-regenerating system (phosphocreatine and creatine phosphokinase). This effect is neither a consequence of a rise in the ATP concentration nor of an increased formation of inorganic phosphate originating from hydrolysis of ATP or phosphocreatine. Addition of ADP to the incubation medium provokes an inhibition of Ca2+ influx and a stimulation of Ca2+ efflux by the microsomal fraction. These results suggest that the stimulation of Ca2+ uptake by the ATP-regenerating system is due, at least in part, to an increase of Ca2+ influx and a slowing down of Ca2+ efflux as consequence of a decrease of ADP availability. It is proposed that the effect of ADP on Ca2+ movements could account for the action of certain agonists on intracellular Ca2+ concentration. Moreover, the InsP3 responsive Ca2+ pool was also shown to be enlarged by the ATP-regenerating system without modification of InsP3 sensitivity.

Adenosine Diphosphate↗
PubMed1996

EGF receptor mRNA and protein in rat lacrimal acinar cells: evidence of its EGF-dependent phosphotyrosilation.

This study was designed to demonstrate the presence of the epidermal growth factor (EGF) receptor in the rat exorbital lacrimal gland. EGF receptor gene transcription was demonstrated 1) by reverse transcription-polymerase chain reaction analysis of lacrimal gland and acinar cells from RNA with a set of specific primers deduced from the rat EGF receptor sequence and 2) by Northern blot analysis of rat lacrimal gland mRNA. Lacrimal acinar cell preparations contain a low but detectable amount of specific 125I-EGF binding sites and efficiently internalize the ligand on binding at 37 degrees C. A sheep polyclonal antibody, directed against the human EGF receptor, detects a protein of 170 kDa by Western blot analysis of membrane proteins of the whole gland. This protein can be immunoprecipitated by the same antibody from whole gland membrane proteins as well as from solubilized acinar cells. Incubations of acinar cells in the presence of EGF results in an increased content of tyrosine-phosphorylated residues in immunoprecipitated 170-kDa protein. Taken together, these results demonstrate for the first time both EGF receptor gene transcription and protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. These results also suggest a specific cellular location of the EGF receptor in a cell population contained in acinar cell preparations.

Animals↗
PubMed1996

Recovery of protein secretion after brefeldin A treatment of rat lacrimal glands: effect of cAMP.

In exocrine cells, the discharge of secretory granule contents in response to extracellular stimuli has been widely documented. However, few data are available concerning the effect of these stimuli on the steps of the secretory pathway preceding protein exocytosis. To obtain more data on this subject, we used brefeldin A (BFA) to perturb intracellular protein transit. When, after exposure of the lacrimal gland lobules to 10 microM BFA, which led to a complete dismantling of the Golgi apparatus and fully inhibited the secretion of newly synthesized proteins, the drug concentration was lowered to 100 nM, a restoration of protein secretion was observed in a secretagogue-dependent manner. Secretagogues increasing the adenosine 3',5'-cyclic monophosphate (cAMP) level facilitated the recovery of protein secretion and Golgi apparatus restructuring, whereas other secretagogues, involving the calcium pathway, did not. Furthermore, the cAMP effect was prevented by H-89, a specific protein kinase A inhibitor. These effects of cAMP are due to neither BFA degradation nor BFA excretion from the cells. We conclude from these results that in rat lacrimal glands the recovery from the dramatic damage caused by BFA is promoted by a cAMP-dependent mechanism and further suggest a role of cAMP in the regulation of the Golgi structure and/or function.

Animals↗
PubMed1995

Protein kinase C and phospholipase D activation in rat parotid glands.

We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.

Alkaloids↗
PubMed1995

Phosphoinositide synthesis in desensitized rat pancreatic acinar cells.

To help understand the possible role of phosphoinositide turnover in the desensitization process, the availability of phosphatidylinositol 4,5-bisphosphate was investigated in normal and desensitized pancreatic acinar cells treated with carbamylcholine (Cch), caerulein (Cae), and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In control acini, incorporation of [myo-3H]inositol into total phosphoinositides was maximal at 120 min, was Cch and Cae dose dependent, and was insensitive to TPA. Cch stimulation increased the proportion of [myo-3H]inositol incorporated into phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], whereas Cae specifically channeled [myo-3H]inositol incorporation into phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. In the desensitized cells, preexposure to Cch and Cae, but not to TPA, increased the subsequent basal rate of [myo-3H]inositol incorporation into total phosphoinositol (PI) by 66 and 50% above control values. There were no subsequent responses to increasing concentrations of Cch, Cae, and TPA during a second incubation. Desensitization of the pancreatic secretory responses to Cch, Cae, and TPA does not seem to result from a decrease either in total PI or in specific PtdIns(4,5)P2 synthesis, which is needed for inositol trisphosphate and diacylglycerol production.

Animals↗
PubMed1995

Effect of microtubule network disturbance by nocodazole and docetaxel (Taxotere) on protein secretion in rat extraorbital lacrimal and parotid glands.

The role of microtubules in the exocrine secretory process is not yet well established. Contradictory effects of anti-microtubule drugs on intracellular transit and protein secretion have been reported. In this work we used microscopic techniques and pulse-chase experiments to compare the involvement of microtubules in the regulated secretory process of two rat exocrine glands: parotid and extraorbital lacrimal glands. In our experiments microtubules were either disrupted by nocodazole or stabilized by a taxoid, docetaxel (Taxotere). We show that the effect of nocodazole and docetaxel on the release of newly synthesized proteins is radically different in the two tissues; in parotid gland they only weakly affect protein release, triggered by stimulation of either muscarinic or beta-adrenergic receptors, but in lacrimal gland, they strongly inhibit protein secretion. This effect or lack of effect of the drug is independent of the signal transduction pathways involved by the different secretagogues used to trigger exocytosis. Furthermore, in lacrimal glands, studies on protein galactosylation (which occurs in the trans-Golgi compartment) indicate that postgalactosylation events are more sensitive to both drugs than pregalactosylation events. On the other hand, we show that the effect of nocodazole and docetaxel on the microtubule network is comparable on the two tissues. Finally, in lacrimal cells, we observed a scattering of the Golgi apparatus concomitant with the disruption of microtubules by nocodazole. We conclude from this study that microtubule network integrity is essential for protein secretion in lacrimal glands but not in parotid glands. This result implies that for the same physiological function, i.e. protein secretion, different mechanisms may be involved.

Animals↗
PubMed1995

[Effect of adrenergic beta-agonist on intracellular free calcium concentration in isolated acini of the parotid gland in rats].

The variations in the intracellular free calcium concentration, [Ca2+]i, have been studied in isolated acini of rat parotid gland with a fluorescent probe, fura-2. In unstimulated acini, [Ca2+]i amounted to a basal value of 180 nM. Isoproterenol used at a concentration of 200 microM, in the presence of a beta-adrenergic blocker, caused a net increase of [Ca2+]i to a mean of 90 nM above basal values. This increase was suppressed by phentolamine, an alpha-adrenergic blocker. A weak increase of [Ca2+]i of 47 nM was also obtained when acini were incubated with 1 microM isoproterenol. This effect was suppressed with 0.1 microM propranolol which, at this concentration, specifically antagonizes the beta-adrenergic receptors. We conclude that isoproterenol at 1 microM, increases the [Ca2+]i by stimulating beta-adrenergic receptors. This effect is not reproduced by forskoline. Our results suggest that this isoproterenol effect on [Ca2+]i is independent of the formation of cyclic AMP. Moreover, when the isoproterenol concentration is high (200 microM), the increase of [Ca2+]i could be through an activation of alpha-adrenergic receptors.

Animals↗
PubMed1994

Are microtubules essential for the secretory process in rat parotid gland?

The role of microtubules in the exocrine secretory process is not yet well established, and their disruption by anti-microtubule drugs leads to variable effects on intracellular transit and protein secretion. We investigated the involvement of microtubules in the regulated secretory process of rat parotid glands using microscopic techniques and pulse-chase experiments. We showed that 10 microM colchicine or nocodazole destroys the microtubule network in parotid acinar cells but only weakly reduces the release of newly synthesized proteins. The half-effect was obtained with 0.22 microM colchicine. Moreover, this small reduction was found to be independent of the nature of the drug (colchicine, colcemid, or nocodazole) and of the nature of the stimulation (beta-adrenergic or cholinergic pathways). Using nocodazole, we have been able to determine that the steps affected by the drug are very early events in the secretory pathway. Finally, we showed by kinetic analysis that microtubule disruption slows protein release only moderately but does not reduce the total amount of secreted protein. We conclude from this study that microtubule integrity is not essential for protein secretion in rat parotid gland.

Animals↗
PubMed1994

Receptor- and phorbol ester-mediated phospholipase D activation in rat parotid involves two different pathways.

We have investigated phospholipase D (PLD) activation in rat parotid acini prelabeled with [14C]stearic acid. In the presence of 2% ethanol, muscarinic and alpha-adrenergic agonists stimulated the formation of [14C]phosphatidylethanol as a result of a PLD activity. The calcium ionophore, ionomycin, and the phorbol esters, 4 beta-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), also stimulated phosphatidylethanol accumulation, but 1-oleyl-2-acetyl-sn-glycerol (OAG), a permeant analogue of diacylglycerol did not. Chelerythrine and staurosporine, two inhibitors of protein kinase C, failed to affect any response. These results suggest that protein kinase C was not involved in the regulation of PLD activity. A difference between PLD regulation by PMA and receptor-mediated agonists was observed with regard to the extracellular calcium requirement. Our results strongly suggest that PLD activation in parotid acini involved different pathways: a calcium-dependent pathway activated by receptor-mediated agonists and a calcium-independent pathway activated by phorbol esters. Moreover, we observed that PLD activation did not result in any change in phosphatidic acid level. We propose that the phosphatidyl transferase activity of PLD reflected a metabolic pathway which may allow a base-exchange reaction in parotid gland.

Adrenergic alpha-Agonists↗
PubMed1994

[Evaluation of locoregional anesthesia in an emergency-traumatology operating room].

Surgical activities concerning traumatic emergencies have been evaluated during 3.5 months. There has been 371 cases. Most of them concerned hand injuries (48%); others concerned limbs, and 5 multiple injuries, one spinal fracture and 30 various injuries. Loco-regional anesthesia has been preferred in 63% of all cases; 80% of all hand injuries, 100% of all femoral neck fractures. The success rate--no addition of morphinics--has been evaluated at 85%, with use of sedatives in 28%.

Adolescent↗
PubMed1993

Phorbol ester-stimulated exocytosis in lacrimal gland: PKC might not be the sole effector.

In this work we show that, although both phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PdBu) stimulate the protein discharge in the rat lacrimal gland with the same half-maximal effective concentration (EC50 approximately 2 x 10(-7) M), PdBu is more efficient in eliciting this response compared with PMA. We also show that sphingosine and chelerythrine have no inhibitory effect on the protein discharge stimulated by PMA or PdBu at concentrations up to 2 x 10(-4) and 3 x 10(-5) M, respectively. With staurosporine, a complete inhibition could not be obtained even at 1 microM. However, only with trifluoperazine (TFP) we obtained a complete inhibition of the PMA-induced protein discharge at 10(-4) M TFP. On the other hand, we show that three diacylglycerol-permeant analogues (1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-didecanoyl-sn-glycerol) do not stimulate protein discharge. In a previous report from our laboratory (30), we showed that the rat lacrimal gland expresses the alpha-isoform of protein kinase C (PKC). In this study, using specific antibodies directed against the newly identified isoforms of PKC, we show on a diethylaminoethyl-cellulose fraction that, besides PKC-alpha, the rat lacrimal gland expresses PKC-epsilon, as previously suggested by Dartt et al. (11), and PKC-delta. Our results question the direct implication of PKC activity as a sole effector of the phorbol ester-stimulated protein secretion in the rat lacrimal gland.

Alkaloids↗
PubMed1993

M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells.

This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.

Animals↗
PubMed1993

Involvement of phospholipase D in caerulein-induced phosphatidylcholine hydrolysis in rat pancreatic acini.

Phosphatidylcholine (PC) metabolism stimulated by caerulein (Cae), a cholecystokinin analogue, was investigated in rat pancreatic acini prelabeled with [3H]choline or [3H]-myristic acid. Both labels were incorporated mostly into PC. An inhibition of choline incorporation into PC was first observed in response to Cae (100 and 500 pM) stimulation, as indicated by reduced [3H]choline incorporation into trichloroacetic acid-precipitable material. Whereas choline incorporation was reduced in PC, Cae (500 pM) significantly increased [3H]choline metabolites release in the incubation medium. Separation of these metabolites by thin-layer chromatography showed that approximately 90% of the labeled products released into the medium was phosphocholine; however, Cae caused significant increases of [3H]choline release after 5, 15, and 30 min. In response to Cae, manoalide, a phospholipase C (PLC) inhibitor, totally prevented phosphocholine release into the medium but did not affect choline release. Staurosporine, a protein kinase C inhibitor, did not influence basal and Cae-induced choline release. In cells prelabeled with [3H]myristic acid, Cae stimulated within 5 min a rapid increase in intracellular [3H]phosphatidic acid (PA) levels in the presence of the PA phosphohydrolase inhibitor, propranolol; this PA production was further increased after 15 and 30 min of stimulation. The time course of [3H]PA formation in the presence of propranolol was similar to that of choline release in the medium. Staurosporine partially blocked PA accumulation stimulated by Cae after 30 min. In contrast, manoalide significantly reduced basal PA accumulation but did not prevent its production in response to Cae. In the presence of ethanol, Cae also significantly stimulated above control values the formation of [3H]phosphatidylethanol. These data indicate that Cae-induced PC hydrolysis in rat pancreatic acini is mediated mostly by phospholipase D (PLD) to produce PA and choline; they suggest a direct action of Cae on PLD activation, an effect independent of PLC activation.

Animals↗
PubMed1993

[Parotid gland in rats, a model for the study of regulated exocrine secretion].

In rat parotid glands protein secretion studied in vitro is weakly stimulated by carbachol (which induces Phospholipase C activation) and strongly by isoprenaline (which activates the adenylate cyclase system). We show in this work that the simultaneous activation of the two types of receptors induces a potentiation of protein secretion. This is not due to an enhanced IP3 or cAMP accumulation nor any modification on calcium movements. Potentiation of protein secretion is also mimicked by analogues of second messengers suggesting that this phenomenon is a post-receptor event which takes place at a distal step from messenger production. Furthermore we also showed that the activation of beta-adrenergic receptor led to two parallel events: cAMP accumulation and calcium movements. These two events were required to obtain maximal secretion. We also show that cholinergic induced secretion is also the result of a synergism between calcium and protein kinase C activation. At a physiological level, the synergism between two different transduction pathways must play an important role. This surely allows the cells to give maximal response, without any desensitization phenomena.

Animals↗
PubMed1992

Evidence for receptor-linked activation of phospholipase D in rat parotid glands. Stimulation by carbamylcholine, PMA and calcium.

In order to test if phospholipase D (PLD) activity exists in the rat parotid gland, we took advantage of the fact that, in the presence of ethanol, PLD generates phosphatidylethanol (PEth) via a transphosphatidylation reaction. Lipid extracts of parotid acini prelabelled with [3H]myristic acid were analyzed by thin layer chromatography to determine [3H]phosphatidylethanol ([3H]PEth) formation. Carbamylcholine (1 mM) stimulated [3H]PEth formation in the presence of 2% ethanol, this effect was completely inhibited by atropine (10 microM). PMA (0.1-1 microM) and ionomycine (10 microM) also caused [3H]PEth generation. We conclude that a phospholipase D activity is present in the rat parotid gland and is regulated by muscarinic cholinergic receptors. Protein kinase C and calcium could also modulate this activity. This report provides the first evidence for the existence and receptor-linked regulation of phospholipase D in an exocrine gland, the rat parotid gland.

Animals↗