Biomedical subjects

L Missiaen

Publications and source records attributed to L Missiaen.

At least 91 records · Page 5Linked to original sources

PubMed1995

Quantal release of calcium in permeabilized A7r5 cells is not caused by intrinsic inactivation of the inositol trisphosphate receptor.

Since the role of intrinsic inactivation of the receptor for inositol 1,4,5-trisphosphate (InsP3) in the phenomenon of quantal release of Ca2+ is still a point of debate, we investigated whether inactivation of the InsP3 receptor can be responsible for quantal release in permeabilized A7r5 cells. A crucial experiment was that a second challenge of the cells with a low dose of InsP3, after washing out the first challenge, was unable to produce a renewed phasic response under conditions where the Ca2+ pumps were blocked. A new phasic response was however obtained with either addition of a higher dose of InsP3 or after allowing the refilling of the Ca2+ stores. We conclude therefore that quantal Ca2+ release in A7r5 cells is not due to InsP3 receptor inactivation.

Animals↗

PubMed1995

Control of inositol 1,4,5-trisphosphate-induced Ca2+ release by cytosolic Ca2+.

The synergistic action of cytosolic Ca2+ and inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ stores has been suggested to be responsible for the complex intracellular Ca2 signals observed during hormonal stimulation of many cell types. However, the ability of cytosolic Ca2+ to potentiate Ca2+ release has recently been questioned because of the observed inhibitory effects of Ca2+ chelators used in previous studies. In the present study, EGTA and BAPTA [1,2-bis-(2-amino-phenoxy)ethane- NNN'N'-tetra-acetic acid] poorly inhibited InsP3-induced Ca2+ release from permeabilized A7r5 smooth-muscle cells. Additionally, stimulatory effects of cytosolic and luminal Ca2+ were observed either in the complete absence of Ca2+ chelator or at constant Ca(2+)-free chelator concentration. These data suggest that potentiation of InsP3-induced Ca2+ release by Ca2+ in A7r5 cells reflects an interaction between Ca2+ and InsP3 receptors, rather than a decrease in chelator-dependent inhibition. The EC50 for activation of InsP3-induced Ca2+ release by cytosolic Ca2+ was unaffected by ATP, or by changing InsP3 concentration, although InsP3-induced Ca2+ release became less sensitive to the inhibitory effects of cytosolic Ca2+ as the InsP3 concentration was elevated. Increasing H+ or Mg2+ concentration shifted the Ca(2+)-activation curve towards higher Ca2+ concentrations. These data suggest that, in addition to the InsP3-binding site, the affinity of the Ca(2+)-binding site(s) on InsP3 receptors can be modulated by intracellular cations.

Adenosine Triphosphate↗

PubMed1995

Slow kinetics of InsP3-induced Ca2+ release: differences between uni- and bi-directional 45Ca2+ fluxes.

The effects of a long-lasting stimulation with inositol 1,4,5-trisphosphate (InsP3) have been studied in monolayers of permeabilized A7r5 cells. When measured under unidirectional 45Ca2+ efflux conditions, i.e. in the presence of 2 microM thapsigargin, an initial fast release was observed which then progressively slowed down into a slow phase which persisted for up to 20 min. When measured under bidirectional 45Ca2+ flux conditions with functional Ca2+ pumps, a transient phase of re-uptake occurred between the initial fast and the subsequent slow release phase. These kinetics are compatible with intrinsic inactivation of the InsP3 receptor. However, this inactivation did not prevent the slow release component. The slow component was not due to the accumulation of an InsP3 metabolite nor to a GTP-dependent translocation of Ca2+ between stores. The slow release phase was more pronounced when the Ca2+ pumps were active than when they were inhibited. This observation is compatible with other findings indicating that the InsP3 receptor is controlled by luminal Ca2+. The decreasing effectiveness of a 20 min lasting InsP3 challenge in mobilizing Ca2+ from less filled stores is most likely due to a progressive depletion of the store and cannot be considered as an experimental artifact caused by a preferential emptying of InsP3-sensitive Ca2+ stores. We conclude that the InsP3 receptor can intrinsically inactivate but that this inactivation is unable to prevent the slow release, which is especially pronounced when Ca2+ pumps are active.

Animals↗

PubMed1995

Rat basophilic leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection.

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP3R-IV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691-21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP3 binding affinity (Kd 3.8 +/- 0.8 nM, Bmax 0.40 +/- 0.08 pmol/mg protein) than microsomes from A7r5 cells (Kd 65 +/- 7 nM, Bmax 0.65 +/- 0.08 pmol/mg protein) or from cerebellum (Kd 135 +/- 14 nM, Bmax 7.35 +/- 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP3R-I was used to determine the presence of the various InsP3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP3R-IV.

Amino Acid Sequence↗

PubMed1994

Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction.

The relative expression of different inositol 1,4,5-trisphosphate receptor (InsP3R) mRNA was determined in a selection of murine and rat cell types that are commonly used to study InsP3-mediated Ca2+ signaling. Different mRNA species (encoding the known InsP3R isoforms) were co-amplified using common polymerase chain reaction primer pairs that recognized sequences that are totally conserved between the various InsP3R. Specific identification of the co-amplified sequences was done by restriction site analysis. In cerebellum, mRNA encoding InsP3R-I accounted for > 90% of the total InsP3R mRNA. This isoform was also present in all other cell types tested and was often the major isoform. In contrast, the level of expression of the other isoforms was cell type-specific. A new InsP3R isoform (type V) was detected that had 94.5% sequence identity with the InsP3R-II in the amplified region. Interestingly, this isoform was largely expressed in murine but not in rat cells. We functionally characterized InsP3R-V using the mouse fibroblast C3H10T1/2 cells, where mRNA encoding InsP3R-V accounted for 76.4% of the total InsP3R mRNA. InsP3-induced Ca2+ release in permeabilized C3H10T1/2 cells was regulated by luminal and cytosolic Ca2+, stimulated by thimerosal, and inhibited by caffeine.

Animals↗

PubMed1994

Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP.

We have investigated the effect of various methylxanthines on the basal and InsP3-stimulated unidirectional 45Ca2+ efflux from permeabilized A7r5 cells under different experimental conditions. We report that caffeine and theophylline inhibit the InsP3-induced Ca2+ release, whereas the basal Ca2+ leak remained largely unaffected. The effect on InsP3-induced Ca2+ release was specific, since isocaffeine was not effective. The inhibition occurred similarly in the absence or presence of extravesicular Ca2+ and was not associated with a decrease in the [3H]InsP3 binding to the receptor. ATP and MgATP (5 mM) prevented the inhibition, suggesting that caffeine may interact with an ATP-binding site on the InsP3 receptor or some associated protein.

Adenosine Triphosphate↗

PubMed1994

Co-activation of inositol trisphosphate-induced Ca2+ release by cytosolic Ca2+ is loading-dependent.

Luminal and cytosolic Ca2+ control the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3). In this work, we have characterized how luminal Ca2+ interfered with the stimulation of the InsP3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. InsP3-induced Ca2+ release from stores containing 34 pmol of Ca2+/10(6) cells absolutely depended on a simultaneous rise in cytosolic Ca2+ concentration in the submicromolar range. This stimulation by cytosolic Ca2+ was more pronounced when the stores were preincubated with Ca2+. In contrast, fully loaded stores containing 3400 pmol of Ca2+/10(6) cells already responded to InsP3 in the absence of cytosolic Ca2+ and the release from these stores was much less stimulated by increasing the cytosolic Ca2+ concentration. The loading dependence of the release was not due to a higher cytosolic Ca2+ concentration around more loaded stores sensitizing the InsP3 receptor by binding at a cytoplasmic site. Luminal Ca2+ was therefore found to functionally substitute for cytosolic Ca2+ in triggering Ca2+ release in the presence of a constant InsP3 concentration. These findings are relevant for explaining base-line Ca2+ spiking in non-excitable cells.

Animals↗

PubMed1994

Kinetics of empty store-activated Ca2+ influx in HeLa cells.

The intracellular Ca2+ indicator Indo-1 was used to monitor changes in cytosolic [Ca2+] ([Ca2+]i) in single HeLa cells upon readmission of external Ca2+ after a short incubation in Ca(2+)-free solution. HeLa cells were responsive to histamine but not to caffeine, and their histamine-sensitive store was totally depleted by a 60-min exposure to 2 microM thapsigargin. The resting [Ca2+]i in thapsigargin-treated cells was higher than in control cells and low amplitude [Ca2+]i oscillations were observed in about 20% of the cells. Readmission of external Ca2+ after a brief withdrawal of extracellular Ca2+ resulted in a transient [Ca2+]i rise, which then decayed to the same elevated [Ca2+]i measured before the Ca2+ withdrawal period. The [Ca2+]i rise was associated with an increased rate of Mn2+ entry, measured as the rate of quenching of intracellular Fura-2. The same procedure did not affect the [Ca2+]i in control cells not pretreated with thapsigargin. The amplitude of this [Ca2+]i transient in thapsigargin pretreated cells depended on the duration of prior incubation in Ca(2+)-free medium. The [Ca2+]i rise induced by elevating the extracellular [Ca2+] from 1.5 to 10 mM was more pronounced if the [Ca2+]i during the initial incubation in 1.5 mM Ca2+ was first lowered by depolarizing the cells. We conclude that an empty store stimulates a Ca2+ entry pathway consisting of two components: a continuously elevated basal leak and a second component that is transient due to the high [Ca2+]i-induced inhibition of the Ca2+ entry pathway. This inhibition and the subsequent recovery from it as the [Ca2+]i is brought to resting levels could cause the oscillatory Ca2+ entry that we recorded in a fraction of the thapsigargin-treated cells.

Biological Transport↗

PubMed1994

Vasopressin responses in electrically coupled A7r5 cells.

Changes in cytosolic Ca2+ concentration ([Ca2+]i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca2+ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca2+]i rise that was about 5 times higher than the amplitude of the spontaneous Ca2+ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca2+ spiking, which persisted after [Ca2+]i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca2+ waves within one cell propagated much more slowly than spontaneous [Ca2+]i rises. We conclude that agonist-induced [Ca2+]i increases in an electrically coupled cell monolayer can be asynchronous.

Animals↗

PubMed1994

Partial calcium release in response to submaximal inositol 1,4,5-trisphosphate receptor activation.

Even a prolonged application of a submaximal dose of Ins(1,4,5)P3 is unable to release the same amount of Ca2+ from the Ins(1,4,5)P3-sensitive store as a higher dose of Ins(1,4,5)P3. Low doses of Ins(1,4,5)P3 therefore only induce a partial release of the stored Ca2+. In this review, we will focus on the mechanisms that may contribute to this behaviour. Molecular heterogeneity of the Ins(1,4,5)P3 receptor can contribute to such behaviour if all the gene products and alternatively spliced isoforms would have different functional properties and be located in different store units. We will show that the control of the Ins(1,4,5)P3 receptor by by luminal Ca2+ also contributes to the partial release behaviour; it can set the sensitivity of the Ins(1,4,5)P3 receptor and the decreasing luminal Ca2+ concentration may inhibit further release while some Ca2+ is still left in the store. It is finally possible that the Ins(1,4,5)P3 receptor may adapt to a maintained stimulus.

Animals↗

PubMed1993

Loading dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in the clonal cell line A7r5. Implications for the mechanism of quantal Ca2+ release.

The Ca2+ content of the intracellular Ca2+ stores controls the inositol 1,4,5-trisphosphate receptor (InsP3R) in the clonal cell line A7r5. This regulation was characterized with respect to the understanding of the "quantal" release phenomenon. Independent of the loading protocol used, increasing the Ca2+ content of the stores increased the sensitivity of the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release until a Ca2+ content of about 30% of the steady-state value was reached. Loading of the stores to higher levels had only a marginal effect on the Ca2+ release. The effects of luminal Ca2+ were still observed in the presence of 10 mM BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), excluding the possibility that luminal Ca2+ acted indirectly via cytoplasmic binding sites. Conditions were developed to simultaneously measure [3H]InsP3 binding to the InsP3R and the 45Ca2+ content of the stores in the same cells. [3H]InsP3 binding to a high affinity binding site was potentiated by luminal Ca2+. Analysis at the molecular level revealed the simultaneous expression of different splice variants of InsP3R-I, as well as the expression of InsP3R-III, and of the putative InsP3R-IV. We conclude that the control of the InsP3R by luminal Ca2+ could account for quantal release and that the observed heterogeneity of the InsP3R may also contribute to this behavior, especially at high levels of store loading.

Animals↗

PubMed1993

Ins(1,4,5)P3 and glutathione increase the passive Ca2+ leak in permeabilized A7r5 cells.

Thapsigargin depletes intracellular Ca2+ stores by its inhibitory effect on the Ca2+ pumps, which unmasks an aspecific Ca2+ leak from the stores. This aspecific Ca2+ permeability of the stores was further investigated using 45Ca2+ fluxes on intact and permeabilized A7r5 smooth-muscle cells. Stores in intact cells were found to be more leaky for Ca2+ than those in saponin-permeabilized or Staphylococcus aureus alpha-toxin-permeabilized cells, which suggests that a cytosolic factor may be involved. Supplementing the medium bathing the permeabilized cells with a submaximal Ins(1,4,5)P3 concentration increased the leakiness of the stores. Glutathione also increased the aspecific Ca2+ leak. This effect occurred with both the reduced and the oxidized form but reduced glutathione was more effective. Our data show that basal Ins(1,4,5)P3 levels and glutathione can contribute to the relatively high Ca2+ leak in intact cells. The washing out of these substances during permeabilization can reduce the aspecific leakiness of the stores.

Bacterial Toxins↗

PubMed1993

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells.

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 microM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of non-mitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 microM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 microM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 microM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca(2+)-loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release.

Arginine Vasopressin↗

PubMed1993

Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 microM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 microM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 microM, and an inhibitory effect at higher concentrations. Thimerosal (2.5-250 microM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 microM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

Animals↗

PubMed1993

Normal Ca2+ signalling in glutathione-depleted and dithiothreitol-treated HeLa cells.

We have investigated whether reducing agents and substances that interfere with glutathione metabolism would affect the histamine-induced rises in internal Ca2+ concentration ([Ca2+]i) in indo-1-loaded HeLa cells. Individual cells responded to 1 microM histamine with either baseline or sinusoidal Ca2+ oscillations, a single Ca2+ peak or a maintained elevation of the [Ca2+]i. Only a few cells did not respond. The sulphydryl reducing agent dithiothreitol (5 mM) did not affect these responses to histamine. A 24-h preincubation with 1 mM DL-buthionine (SR)-sulphoximine, which reduces the cellular glutathione content to less than 20% of its control value, affected neither these histamine responses, nor the [Ca2+]i rises after application of 2 microM thapsigargin. We conclude that oxidation of critical sulphydryl groups is not required for the normal response to histamine and also that glutathione plays no role in agonist-induced Ca2+ signalling in HeLa cells.

Antimetabolites↗

PubMed1993

[Ca2+ homeostasis in mammalian cells].

The cytoplasmic free calcium concentration ([Ca2+]i) is one of the most important factors in the regulation of the cellular activity. We therefore studied in this work the changes of [Ca2+]i during muscle excitation with optical techniques in intact smooth muscle preparations. Also the relation between [Ca2+]i, the phosphorylation of the myosin light chain and the force development in phasic and tonic smooth muscle during excitation-contraction coupling was investigated. We concluded that the changes of [Ca2+]i and force are not invariably tightly coupled during stimulation in spite of the fact that the increase of [Ca2+]i is the primary regulator of smooth muscle contraction. Agonists may, besides their Ca2+ mobilizing effect, also modulate the sensitivity of the regulatory-contractile apparatus for Ca2+. Ca(2+)-release from the internal stores during activation may modulate its further depletion. We demonstrated that the Ins(1,4,5)P3 receptor is controlled by luminal Ca2+. These observations allowed us to develop a model whereby Ca2+ oscillations during submaximal stimulation could be explained. This model of control by luminal Ca2+ also explained why the release in response to a low Ins(1,4,5)P3 concentration suddenly stops at a time when there is still plenty of Ca2+ in the internal stores. We furthermore showed that these Ca2+ channels in the SR are controlled by the redox-status of the cell. The study of Ca2+ extrusion was mainly focused on the Ca2+ pump of the plasma-membrane and its regulation by negatively charged phospholipids. Our main finding was that the plasma-membrane Ca2+ pump was stimulated by negatively charged phospholipids. Finally, we started the investigation of the existence of Ca2+ gradients between nucleus and cytoplasm in intact cells with confocal laser microscopy. We demonstrated in different cell types the existence of spatio-temporal differences in the Ca2+ distribution between nucleus and cytosol in basal and stimulated conditions. These observations indicate that a Ca2+ barrier must be present at the nuclear envelope, that different Ca2+ mobilizing phenomena are present and are in favour of the existence of a specific regulation of nuclear Ca2+.

Animals↗

PubMed1992

2,5-Di-(tert-butyl)-1,4-benzohydroquinone and cyclopiazonic acid decrease the Ca2+ permeability of endoplasmic reticulum.

Specific inhibitors of the endoplasmic-reticulum Ca2+ pump will deplete intracellular stores and are therefore useful to study the role of store depletion on plasma-membrane Ca2+ permeability. We now report that the Ca(2+)-pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) reduces the passive Ca2+ leak from the internal stores in permeabilized A7r5 vascular smooth-muscle cells. This aspecific effect occurred at concentrations that are normally used to empty the stores in intact cells. Cyclopiazonic acid exerted a similar, although less pronounced effect, while thapsigargin did not affect the passive Ca2+ leak. The inositol 1,4,5-trisphosphate-mediated Ca2+ release was not affected. tBuBHQ and cyclopiazonic acid cannot therefore be used as specific tools to probe the mechanism of receptor-mediated Ca2+ entry.

Benzoquinones↗

PubMed1992

Ca(2+)-transport ATPases and their regulation in muscle and brain.

Eukaryotic cells express one or more isoforms of a sarco(endo)plasmic reticulum (SERCA) and of a plasma membrane (PMCA) Ca2+ pump. Both the SERCA and PMCA gene transcripts are subject to alternative processing in a differentiation stage-dependent and tissue-dependent manner. The Ca2+ pump isoforms thus generated may present different functional properties. This is exemplified by the SERCA2a and SERCA2b isoforms which differ in their Ca2+ sensitivity. Analysis of the cDNA structures for PMCA1 predicts protein isoforms with variant calmodulin- and phospholipid-binding domains. A comparative study of the tissue-specific mechanisms governing SERCA-PMCA transcript processing and a more detailed study of the functional implication of the PMCA pumps isoform diversity will be challenging subjects for future studies.

Alternative Splicing↗