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R Casteels

Publications and source records attributed to R Casteels.

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

cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 gene.

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5' region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3'-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5'-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177-6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

Amino Acid Sequence↗
PubMed1996

Kinetics of the non-specific calcium leak from non-mitochondrial calcium stores in permeabilized A7r5 cells.

We have investigated the detailed kinetics of the passive Ca2+ leak from non-mitochondrial Ca2+ stores in permeabilized A7r5 cells. The decrease in the content of stored Ca2+ in the presence of 2 microM thapsigargin deviated from a single-exponential curve in the initial phase of the efflux. The deviation persisted after correcting this efflux for passively bound Ca2+. The non-single-exponential nature of the spontaneous release also occurred when the initial store Ca2+ content was reduced to 40% of its original value by pretreatment with 200 nM inositol 1,4,5-trisphosphate (InsP3). The passive Ca2+ leak could be modelled by two exponential curves with discrete rate constants of 0.06 min-1 and 0.98 min-1, and not by any other type of non-exponential decay. We concluded that individual store units are heterogeneous with respect to their passive Ca2+ permeability. This non-exponential nature of the passive Ca2+ release is unrelated to the non-single-exponential InsP3-induced Ca2+ release.

Animals↗
PubMed1996

Threshold for inositol 1,4,5-trisphosphate action.

We developed a unidirectional 45Ca2+ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3 action, which was around 32 nM InsP3 under control conditions. The InsP3-induced Ca2+ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP3 concentrations. Stimulatory concentrations of thimerosal (10 microM) shifted the threshold to 4.5 nM InsP3 and increased both the cooperativity and the maximal normalized rate of Ca2+ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 microM) but then the effects on the cooperativity and on the normalized rate of Ca2+ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP3 concentrations. Luminal Ca2+ did not affect the threshold for InsP3 action but stimulated the normalized release at each InsP3 concentration. The inhibitory effect of 10 microM free cytosolic Ca2+ was associated with a shift in threshold to higher InsP3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP3 receptor.

Adenosine Triphosphate↗
PubMed1996

Hypotonically induced calcium release from intracellular calcium stores.

Osmotic cell swelling induced by hypotonic stress is associated with a rise in intracellular Ca2+ concentration, which is at least partly due to a release of Ca2+ from internal stores. Since osmotic influx of water dilutes the cytoplasmic milieu, we have investigated how nonmitochondrial Ca2+ stores in permeabilized A7r5 cells respond to a reduction in cytoplasmic tonicity. We now present experimental evidence for a direct Ca2+ release from the stores when exposed to a hypotonic medium. The release is graded, but does not occur through the inositol trisphosphate or the ryanodine receptor. Ca2+ seems to be released through the passive leak pathway, and this phenomenon can be partially inhibited by divalent cations in the following order of potency: Ni2+ = Co2+ > Mn2+ > Mg2+ > Ba2+. This release also occurs in intact A7r5 cells. This novel mechanism of hypotonically induced Ca2+ release is therefore an inherent property of the stores, which can occur in the absence of second messengers. Intracellular stores can therefore act as osmosensors.

Animals↗
PubMed1996

Initiation sites for Ca2+ signals in endothelial cells.

Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca2+ concentration and rate of wave propagation than the cell body. Ca2+ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca2+ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca2+ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca2+ release in the individual store units to produce a Ca2+ wave.

Animals↗
PubMed1996

Mechanisms responsible for quantal Ca2+ release from inositol trisphosphate-sensitive calcium stores.

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP3) and, after activation of the InsP3 receptor (InsP3R), to Ca2+ release from intracellular Ca2+ stores. The release of intracellular Ca2+ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called "quantal Ca2+ release", is that even the maintained presence of a submaximal dose of agonist or of InsP3 for long time periods (up to 20 min) provokes only a partial release of Ca2+. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP3-induced Ca2+ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca2+ stores to function as increment detectors and to induce local Ca2+ responses. The basic mechanism for quantal release of Ca2+ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP3- induced Ca2+ release include the presence of InsP3Rs with varying sensitivities for InsP3, heterogeneous InsP3R distribution, intrinsic inactivation of the InsP3Rs, and regulation of the InsP3Rs by Ca2+ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca2+-mediated conformational change of the InsP3R is most likely the key feature of the mechanism for quantal Ca2+ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.

Animals↗
PubMed1996

The effects of ketamine on Ca2+ movements in A7r5 vascular smooth muscle cells.

To investigate the effects of ketamine on Ca2+ movement to and from intracellular Ca2+ stores and across plasma membranes, 45Ca2+ fluxes were studied in permeabilized and intact A7r5 smooth muscle cells, an established cell line derived from embryonic rat aorta. Monolayers of A7r5 cells were loaded with 45Ca2+, and the radioactivity in the collected medium and the residual activity were measured by liquid scintillation counting. Ketamine had no effect on 45Ca2+ uptake and passive leak of the nonmitochondrial Ca2+ pool in permeabilized A7r5 cells. Ketamine 1 mM had no inhibitory effect on the inositol 1,4,5-trisphosphate (InsP3, 1 microM)-induced Ca2+ release from the intracellular stores. In intact A7r5 cells, ketamine did not alter the Ca2+ extrusion from these cells under resting conditions. Addition of 10 nM vasopressin resulted in a transient Ca2+ release from the intracellular stores. Ketamine inhibited this vasopressin-induced Ca2+ release, but did not enhance Ca2+ extrusion through the plasma membrane in the period after the vasopressin effect. These results indicate that ketamine inhibits agonist-induced Ca2+ release from intracellular stores, but has no effect on Ca(2+)-uptake into intracellular stores or on Ca2+ extrusion through the plasma membrane in A7r5 smooth muscle cells.

Anesthetics, Dissociative↗
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

Human cytomegalovirus modulates the Ca2+ response to vasopressin and ATP in fibroblast cultures.

The free calcium concentrations in the nucleus ([Ca2+]n) and in cytosol ([Ca2+]c) of cultured human embryonic lung (HEL) fibroblasts were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. In resting HEL cells, The free cellular Ca(2+)-concentration significantly increased upon human cytomegalovirus (HCMV) infection. The ratio between [Ca2+]n and [Ca2+]c was not affected. Following stimulation by ATP or [Arg8] vasopressin (AVP), a differential Ca2+ response of the HCMV-infected HEL cells was observed. While uninfected cells were highly sensitive to AVP and only poorly sensitive to ATP, infected cells showed a high responsiveness to ATP but not to AVP. This switch in sensitivity to the agonists first observed at 24 h post infection. The Ca(2+)-rise following ATP or AVP stimulation was derived from intracellular Ca2+ stores. The magnitude of the ATP-induced Ca(2+)-rise increased upon infection. In contrast to non-infected cells where [Ca2+]n > [Ca2+]c during stimulation with AVP or ATP, no nucleo-cytosolic Ca(2+)-gradient was observed in infected cells. Furthermore, the magnitude of the Ca2+ rise in the two compartments was higher in ATP-stimulated cells. It is concluded that HCMV infection significantly interferes with Ca(2+)-homeostasis in HEL cells which could be related to the pathogenesis of the disease.

Adenosine Triphosphate↗
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

The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca(2+)-transport ATPase (SERCA2a/b).

Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains in the SERCA2b tail were analysed by constructing three SERCA2b deletion mutants lacking 12, 31 or 49 amino acids. The mutants and the parental SERCA2 pumps were expressed in COS-1 cells and analysed for functional difference. SERCA2b had a twofold higher Ca2+ affinity, a twofold lower turnover rate and a 10-fold lower vanadate-sensitivity than SERCA2a and the mutants. Since each of the three truncated versions of SERCA2b acquire the characteristic properties of SERCA2a, it is concluded that the stretch of the last 12 residues of SERCA2b is of critical importance.

Amino Acid Sequence↗
PubMed1994

Regulation of splicing is responsible for the expression of the muscle-specific 2a isoform of the sarco/endoplasmic-reticulum Ca(2+)-ATPase.

Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non-muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site located between the donor and acceptor splice site of an intron which is only removed in muscle tissues. To define the cis-active elements involved in differential processing, we constructed a minigene (pCM beta SERCA2) containing the 3' end of the SERCA2 gene. When stably transfected into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This proves that the essential elements required for regulated processing are present in the construct. Furthermore, co-transfection of the pCM beta SERCA2 minigene and a myogenin expression vector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SERCA2. This shows that trans-acting factor(s) responsible for muscle-specific processing can be induced by one of the important regulatory genes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simple competition model between splicing and polyadenylation, but it is consistent with splicing being very inefficient in non-muscle cells. Moreover, splicing could be induced in non-muscle cells by optimizing the muscle-specific donor splice site and/or by shortening the intron length. We therefore propose that expression of the muscle-specific SERCA2a isoform is the result of activation of an otherwise inefficient splicing process.

Animals↗
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↗