Biomedical subjects

C Randriamampita

Publications and source records attributed to C Randriamampita.

16 recordsLinked to original sources

PubMed2004

Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling".

Interaction between a T cell and an antigen-presenting cell leads to the rapid formation of an immunological synapse allowing antigen detection by the T cell and the development of an immune response. Antigen detection triggers various cellular responses including a modest but sustained T cell Ca2+ increase. In this review are discussed a series of related questions. What are the various molecular events by which a T cell Ca2+ response can be triggered in the immunological synapse by a very small amount of antigen? How is Ca2+ released from intracellular stores and how can these stores remain empty for hours? Through which channels does Ca2+ influx takes place, and how is Ca2+ influx coupled to Ca2+ release from intracellular stores? What are the main immediate and indirect cellular targets of the Ca2+ increase?

Animals↗

PubMed2000

Rac is involved in early TCR signaling.

The GTPase Rac controls signaling pathways often related to actin polymerization in various cell types. In T lymphocytes, Rac is activated by Vav, a major component of the multiprotein transduction complex associated to the TCR. Although profound signaling defects have been observed in Vav-deficient mice, a role of Rac in the corresponding early TCR signaling has not been tested directly. This question was investigated in Jurkat T cells transfected with either a dominant-negative (RacN17) or a constitutively active (RacV12) form of Rac. In T cells expressing either RacN17 or RacV12, the anti-CD3-induced Ca2+ response and production of inositol-1,4,5-trisphosphate were inhibited. The basal level of phosphatidylinositol-4,5-bisphosphate was not significantly diminished by Rac mutants. The major inhibitory effect of Rac mutants on Ca2+ signaling is exerted on the activity of phospholipase C-gamma and, before that, on the phosphorylation of ZAP-70 and of the linker molecule for activation of T cells, LAT. An anti-CD3-induced increase in actin polymerization was observed in control cells but not in cells transfected with a Rac mutant. In addition, latrunculin, which binds to monomeric actin, simultaneously inhibited basal and CD3-induced actin polymerization and Ca2+ signaling. These findings suggest a link between the effects exerted by Rac mutants on cortical actin polymerization and on TCR signaling. Rac cycling between its GTP- and GDP-bound states is necessary for this signaling. Alterations observed in early TCR-dependent signals suggest that Rac contributes to the assembly of the TCR-associated multiprotein transduction complex.

Actins↗

PubMed2000

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry.

BACKGROUND: The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. METHODS: We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry. RESULTS: With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.

ADP-ribosyl Cyclase↗

PubMed2000

The invasion protein InIB from Listeria monocytogenes activates PLC-gamma1 downstream from PI 3-kinase.

Entry of the bacterial pathogen Listeria monocytogenes into non-phagocytic mammalian cells is mainly mediated by the InlB protein. Here we show that in the human epithelial cell line HEp-2, the invasion protein InlB activates sequentially a p85beta-p110 class I(A) PI 3-kinase and the phospholipase C-gamma1 (PLC-gamma1) without detectable tyrosine phosphorylation of PLC-gamma1. Purified InlB stimulates association of PLC-gamma1 with one or more tyrosine-phosphorylated proteins, followed by a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels and a release of intracellular Ca2+ in a PI 3-kinase-dependent manner. Infection of HEp-2 cells with wild-type L. monocytogenes bacteria also induces association of PLC-gamma1 with phosphotyrosyl proteins. This interaction is undetectable upon infection with a deltainlB mutant revealing an InlB specific signal. Interestingly, pharmacological or genetic inactivation of PLC-gamma1 does not significantly affect InlB-mediated bacterial uptake, suggesting that InlB-mediated PLC-gamma1 activation and calcium mobilization are involved in post-internalization steps.

Bacterial Proteins↗

PubMed1995

Degradation of a calcium influx factor (CIF) can be blocked by phosphatase inhibitors or chelation of Ca2+.

In many cell types, depletion of Ca2+ stores causes activation of Ca2+ influx by a mechanism whose molecular basis remains unclear. We recently described a new messenger that is released by empty Ca2+ stores and that activates Ca2+ influx in heterologous cells (Randriamampita, C. & Tsien, R. Y. (1993) Nature 364, 809-814). This factor, provisionally named CIF (for Ca2+ influx factor), seems to be a small nonprotein factor possessing a phosphate group. Meanwhile Parekh et al. reported that okadaic acid, an inhibitor of protein phosphatases 1 and 2A, potentiates Ca2+ influx in Xenopus oocytes (Parekh, A. B., Terlau, H. & Stühmer, W. (1993) Nature 364, 814-818). A link between these two observations is presented in this paper. We show that in astrocytoma cells, okadaic acid and cyclosporin A (an inhibitor of calcineurin) both potentiate the Ca2+ elevations due to low doses of CIF, thapsigargin, or carbachol. In lymphocytes, okadaic acid potentiates the Ca2+ elevations due to low doses of phytohemagglutinin and increases the amount of extractable CIF. CIF degradation can be observed in cell-free homogenates of lymphocytes and is prevented by the above phosphatase inhibitors, an effect that can at least partly explain their potentiation of Ca2+ influx. CIF degradation is also prevented by lowering free Ca2+ concentrations, which could be a feedback mechanism to enhance Ca2+ influx when cells are depleted of Ca2+.

Astrocytes↗

PubMed1994

Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells.

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.

Calcium↗

PubMed1993

Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx.

Intracellular Ca2+ signals that last more than a few minutes after the onset of stimulation depend critically on influx of extracellular Ca2+. Such Ca2+ influx can be triggered in many cell types by depletion of intracellular Ca2+ stores without detectable elevations of known messengers. The mechanism by which store depletion can control plasma membrane Ca2+ permeability remains controversial. Here we present evidence for a novel soluble mediator. Calcium depletion of a lymphocyte cell line caused the messenger to be released from intracellular organelles into the cytoplasm and to a much lesser extent into the extracellular medium. The messenger caused Ca2+ influx when applied to macrophages, astrocytoma cells, and fibroblasts and was therefore named CIF (for Ca(2+)-influx factor). CIF appears to have hydroxyls (or hydroxyl and amino groups) on adjacent carbons, a phosphate, and a M(r) under 500.

Astrocytoma↗

PubMed1991

Cyclic-GMP-dependent refilling of calcium stores in macrophages.

The effects of cyclic GMP on the release of calcium from intracellular stores, induced in murine peritoneal macrophages by either ATP or platelet-activating factor, were studied by microfluorimetry with fura-2. When macrophages were incubated for 10-20 min with 10 microM LY83583, an inhibitor of guanylate cyclase, the rise in intracellular calcium induced by agonist application was strongly depressed. This inhibition of the response to platelet-activating factor could be reversed by the addition of 0.1 mM cyclic 8-bromo-GMP. In the presence of cyclic 8-bromo-GMP, the decay of the calcium transient was speeded. Furthermore, when two calcium transients were evoked within 1 min by stimulating the cells with 10 microM ATP, the second calcium transient was more depressed than the first one in the presence of LY83583. These findings are compatible with the hypothesis that cyclic GMP is necessary for the activation of the calcium pump of the intracellular stores.

Adenosine Triphosphate↗

PubMed1991

Nitrendipine-induced inhibition of calcium influx in a human T-cell clone: role of cell depolarization.

An increase in intracellular calcium concentration stimulated by anti-CD2 or CD3 antibodies has been measured with Fura-2 in P28 cells, a human CD4+ T cell clone. This intracellular calcium increase was sensitive to membrane potential changes, being increased when the cells were hyperpolarized and decreased when they were depolarized. The intracellular calcium increase was inhibited by nitrendipine (1-50 microM). Nitrendipine also induced a depolarization of the cells, due to the blockade of a potassium conductance. The inhibition of the calcium increase caused by nitrendipine could be partially reversed by hyperpolarizing the cells with valinomycin. It is concluded that the effects of nitrendipine on potassium channels may account for a large part of the inhibition that nitrendipine exerts on the calcium increase elicited by CD2 or CD3 stimulation.

Antibodies, Monoclonal↗

PubMed1991

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.

Animals↗

PubMed1990

Arachidonic acid activates Ca2+ extrusion in macrophages.

Stimulation of macrophages with platelet-activating factor (PAF) elicits an increase of intracellular calcium concentration, Ca2+i, which was monitored here at the single cell level with the calcium-sensitive dye Fura-2. The sustained component of this Ca2+i increase reflects the dynamic balance achieved between enhanced Ca2+ influx and efflux. In macrophages where a steady increase of Ca2+i has been evoked by 50 nM thapsigargin (a molecule known to empty Ca2+ stores and elevate Ca2+i in various cell types), PAF activates Ca2+ efflux, without causing a preceding increase in Ca2+i. This result shows that in this case, Ca2+ extrusion is not merely a consequence of a Ca2+i increase. PAF-evoked Ca2+ extrusion does not result from the activation of the Na+/Ca2+ exchanger. Exogenous arachidonic acid (10-100 microM) elicits Ca2+ efflux in macrophages where Ca2+i has been previously elevated by either PAF or thapsigargin. PAF-induced Ca2+ extrusion is blocked by 4-bromophenacylbromide, an inhibitor of arachidonic acid production by phospholipase A2. Together, these results suggest that arachidonic acid, which is produced in PAF-stimulated macrophages, contributes to the regulation of a Ca2+ extrusion system, which is presumably a Ca2(+)-ATPase.

Animals↗

PubMed1989

Biphasic increase in intracellular calcium induced by platelet-activating factor in macrophages.

In single mouse macrophages stimulated by platelet-activating factor (PAF), the intracellular calcium concentration (Cai) monitored with fura-2 at room temperature presents a biphasic increase, including a transient and a more sustained component. After pulse administration of PAF, the first phase lasts for a few seconds and reaches a peak value of 0.5-1 microM Ca2+ at high PAF concentration. The amplitude of this peak is independent of extracellular Ca2+ concentration, suggesting that the initial Ca2+ transient is due to the release of Ca2+ from intracellular stores. The second phase of the response lasts for several minutes; its maximum amplitude is reached 1-2 min after the brief initial PAF stimulation. This phase, suppressed in zero external Ca2+ and increased in 10 mM Ca2+, is probably due to influx of Ca2+ through the plasma membrane. This secondary Ca2+ increase is blocked by 10-50 microM lanthanum. At low PAF concentration, the initial Ca2+ transient is not followed by a second phase, showing that the initial rises of Ca2+ and of its activator (presumably inositol trisphosphate) are not sufficient to trigger the second phase of Ca2+ increase.

Animals↗

PubMed1989

Arachidonic acid closes gap junction channels in rat lacrimal glands.

The effects of arachidonic acid (AA) on gap junction conductance of rat lacrimal glands have been studied with the double patch-clamp technique. Extracellular application of 50-100 microM AA for a few minutes induced a closure of gap junction channels. This effect was mimicked by linoleic acid and by other non-degradable fatty acids (myristic and lauric), and was not blocked by inhibitors of AA metabolism. This suggests that the active molecule was the fatty acid itself, and not one of its oxidative derivatives. Inhibitors of AA metabolism caused a certain degree of uncoupling by themselves, probably due to the accumulation of AA. This effect was reduced in the presence of 10 microM 4-bromophenacylbromide, an inhibitor of phospholipase A2. The effect of AA did not seem to be mediated by an increase of intracellular Ca concentration, nor by a change in the activity of guanylate or adenylate cyclases, nor by activation of protein kinase C. Therefore it could be attributed to a direct effect of AA on gap junctions. Analysis of single gap junction channel currents showed that AA-induced closure of these channels resulted from a change in the number of open channels, and not from a reduction of their conductance. Finally, acetylcholine-induced closure of gap junction channels was not primarily mediated by an increase in AA concentration, although such an increase may well follow the activation of muscarinic receptors and play a role in the acetylcholine effect.

Acetophenones↗

PubMed1988

Calcium and secretagogues-induced conductances in rat exocrine pancreas.

The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K were PCl/PK approximately equal to 3. At 1 microM internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 microM internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.

Acetylcholine↗

PubMed1988

Acetylcholine-induced closure of gap junction channels in rat lacrimal glands is probably mediated by protein kinase C.

In rat lacrimal gland cells, application of acetylcholine (ACh) opens Ca-dependent channels and closes gap junction channels. We have shown previously that the increase in intracellular calcium concentration induced by ACh, is not required for the closure of gap junctions. We have examined the effects of activators of protein kinase C on gap junction conductance measured with the double patch-clamp technique. This conductance was markedly reduced by incubating the cell pairs for a few minutes with 100 nM phorbol dibutyrate (PdBu). Two membrane permeant analogues of diacylglycerol, OAG (1-oleoyl-2-acetyl-glycerol) and DOG (1,2-dioleoyl-glycerol) also induced a closure of gap junction channels. This effect was observed in the concentration range 10-100 microM when the diacylglycerol was added on intact cells, and at 75 microM when it was applied on dialysed cells. The cell uncoupling was not mediated by phosphatidate, a degradation product of OAG, nor by a phospholipase A2-induced increase in arachidonate concentration. The OAG-induced closure of gap junction channels reversed spontaneously upon prolonged exposure (more than 90 min at 37 degrees C) to 25 microM OAG. After a prolonged OAG treatment, the ability of ACh to uncouple the cells was markedly reduced. ACh induced uncoupling was modulated to some extent by intracellular Ca and had an absolute requirement for Mg. These results indicate that ACh-induced closure of gap junction channels may be mediated by PKC.

Acetylcholine↗

PubMed1987

Ionic channels in murine macrophages.

In this paper we examine the different voltage or calcium-dependent currents present in murine peritoneal macrophages, and in a macrophage-like cell line, J774. Three of these are K currents while the fourth is carried by Cl. One K current, activated by hyperpolarization, has all the characteristics of the inward rectifier found in egg or muscle cells. It appears in peritoneal macrophages only after several days in culture. A second K current, activated by depolarization, is a typical delayed rectifier. The amplitude of these currents and, as a consequence, the membrane potential of the cells, can be markedly changed by the movement of fluid around the cells. A third K current is activated by internal calcium levels in the micromolar range. It presents a low-voltage sensitivity and is blocked by 0.1-1 mM quinine. The Cl current flows through large-size channels (180-390 pS) that are active mainly in excised patches. These channels are unlikely to be half gap junctional channels, as suggested in former studies. The second goal of this paper is to examine if the activation of receptors for the Fc fragment of IgGs (Fc receptors) is associated with a change in the electrical properties of the membrane of macrophages. We have observed that the binding of multivalent ligands (the monoclonal antibody 2.4G2, aggregated IgGs, or sheep red blood cells coated with IgGs) to their Fc receptors on adherent macrophages did not trigger any change in resting potential. This is a surprising difference with former results obtained on non-adherent J774 cells (Young, J. D.-E., J. C. Unkeless, H. R. Kaback, and Z. A. Cohn, 1983, Proc. Natl. Acad. Sci. USA., 80:1357-1361) and on human alveolar macrophages (Nelson, D. J., E. R. Jacobs, J. M. Tang, J. M. Zeller and R. C. Bone, 1985, J. Clin. Invest., 76:500-507).

Animals↗