Search PubMed⌕ Search

Biomedical subjects

L Missiaen

Publications and source records attributed to L Missiaen.

At least 73 records · Page 4Linked to original sources
PubMed1997

Slow kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: is the release 'quantal' or 'non-quantal'?

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores is generally assumed to be a 'quantal' process because low InsP3 concentrations mobilize less Ca2+ than high concentrations and a submaximal concentration does not release all the InsP3-mobilizable Ca2+. However, some recent reports questioned the generally accepted view that a low dose of InsP3 is unable to empty the whole store. We have now challenged the stores of permeabilized A7r5 cells in InsP3 for much longer periods than previously reported, to assess directly whether the slow phase of the release would empty the whole store (a non-quantal response) or only a fraction of it (a quantal response). Addition of a maximal [InsP3] at the end of a prolonged (92 min) stimulation with a submaximal [InsP3] resulted in additional Ca2+ release. Experiments in which the stores were challenged with different submaximal InsP3 concentrations for long time periods revealed that a lower [InsP3] never released the same amount of Ca2+ as a higher [InsP3]. This quantal pattern of Ca2+ release occurred both at 25 degrees C and at 4 degrees C. There was a time-dependent increase in the fraction of Ca2+ recruited by the lower compared with the higher [InsP3]. This recruitment of Ca2+ persisted if the [InsP3] was decreased, but was largely prevented by palmitoyl-CoA, a potent blocker of the luminal Ca2+ translocation between individual store units. ATP, in the presence of InsP3, released Ca2+ under conditions permitting the recruitment of no additional InsP3 receptors, indicating that an all-or-none emptying of a fraction of the stores cannot be the only mechanism responsible for quantal Ca2+ release in A7r5 cells. We conclude that some of the previously published evidence for a non-quantal Ca2+ release pattern should be reinterpreted.

Adenosine Triphosphate↗
PubMed1997

Isoform diversity of the inositol trisphosphate receptor in cell types of mouse origin.

Previous reports suggested the expression of four or five different Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] isoforms in mouse cells [Ross, Danoff, Schell, Snyder and Ullrich (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4265-4269; De Smedt, Missiaen, Parys, Bootman, Mertens, Van Den Bosch and Casteels (1994) J. Biol. Chem. 269, 21691-21698]. To explore this diversity further, we have isolated and sequenced partial clones of two Ins(1,4,5)P3R mRNAs from the mouse embryonic C3H10T1/2 cell line. These clones showed between 94.2 and 94.9% sequence identity with the corresponding rat Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms. Based on these newly obtained sequences we have determined the relative expression of the different Ins(1,4,5)P3R mRNAs in cultured cells and in animal tissues of mouse origin by a ratio reverse transcriptase polymerase chain reaction (RT-PCR). Ins(1,4,5)P3R-I was very prominent in brain and cerebellum and Ins(1,4,5)P3R-II in epithelia such as kidney as well as in both cardiac and skeletal muscle. Ins(1,4,5)P3R-III was highly expressed in all cultured cell types and in tissues with high cell turnover, e.g. testis. The prominent expression of Ins(1,4,5)P3R-I and Ins(1,4,5)P3R-III in A7r5 and C3H10T1/2 cells respectively was confirmed by immunoblot analysis and was compatible with a lower threshold for Ins(1,4,5)P3-induced Ca2+ release in the former cell type. Screening of a large number of mouse cell lines and tissues revealed the presence of Ins(1,4,5)P3R-I as well as of the Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms which were identified in the present study, but in contrast with previous reports there was no evidence for more isoform diversity.

Animals↗
PubMed1997

Synergism between hypotonically induced calcium release and fatty acyl-CoA esters induced calcium release from intracellular stores.

The non-mitochondrial Ca2+ stores in permeabilized A7r5 cells responded to a decrease in Mg-ATP concentration with a pronounced Ca2+ release if 20 microM CoA was present. This release was rather specific for the preincubation or removal of ATP. ATP gamma S was much less effective and AMP-PNP, GTP, ITP, CTP, UTP, ADP, AMP, adenosine and adenine had no effect. CoA activated with an EC50 of 6 microM. Dephospho-CoA was a less effective cofactor and desulfo-CoA was ineffective. The release induced by Mg-ATP removal did not occur in the presence of 2% fatty acid-free bovine serum albumin and did not develop at 4 degrees C. All these findings suggest that CoA had to be acylated by endogenous fatty-acyl-CoA synthetase to become effective. Myristoyl- and palmitoyl-CoA esters were identified as the most effective cofactors for the release. Ca2+ release induced by removing Mg-ATP did not occur if the osmolality of the medium was kept constant by addition of mannitol, sucrose, KCl, MgCl2 or Mg-GTP, indicating that the decrease in tonicity was the trigger for the release. Mg-ATP plus CoA also synergized with Ca2+ release induced by a hypotonic shock imposed by diluting the medium with H2O. Osmolality changes induced by decreasing the Mg-ATP concentration were more effective in releasing Ca2+ than equal decreases in concentration of all solutes. We conclude that fatty acyl-CoA esters sensitize the hypotonically induced Ca2+ release from the non-mitochondrial Ca2+ stores.

Acyl Coenzyme A↗
PubMed1997

Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones.

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.

Animals↗
PubMed1997

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2+ binding proteins in RBL-2H3 rat basophilic leukemia cells.

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca2+ binding proteins in fraction F3 and the sucrose density gradient fractions. InsP3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCAs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.

Animals↗
PubMed1996

Distribution of the organellar Ca2+ transport ATPase SERCA2 isoforms in the cat brain.

Of the three genes encoding the Ca2+ transport ATPases of the endoplasmic reticulum, the SERCA2 gene is the major isoform expressed in the mammalian brain. The SERCA2 transcript is alternatively processed generating two protein isoforms: SERCA2a which is expressed in cardiac and slow-skeletal muscle, and SERCA2b, the house-keeping isoform which is ubiquitously expressed. We have studied the expression of SERCA2 in the cat brain, and at a less refined level also in the rat brain, using antibodies specific for either SERCA2a or SERCA2b. The SERCA2a staining was very restricted. The SERCA2a antibody clearly labeled the cell body of the Purkinje neurons and weakly stained the giant cells of the gigantocellular reticular nuclei. In contrast, the SERCA2b isoform was found in most regions of the brain. It appeared to be largely confined to neuronal cells. Neuroglial cells were negative. The antibody stained the cell body. In heavily labeled cells such as the pyramidal cells of the hippocampus and of the cerebral cortex, it also stained the proximal portion of the dendrites. The most intense labeling was observed in the Purkinje neurons, which were stained all over the cell including the distal ramifications of the dendritic tree. Remarkably the SERCA2b labeling in neuronal cells of the hypothalamic area and the substantia nigra was very weak. The possible physiological significance of these results is discussed.

Animals↗
PubMed1996

Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5-trisphosphate receptor.

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP3R as glutathione S-transferase fusion proteins. 45Ca2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site. The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. A K0.5 of 0.3 microM and a Hill coefficient of 1.1 were determined by 45Ca2+ overlay by quantification of 45Ca2+ binding on blots. The cytosolic Ca2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca2+ binding was mapped to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+, with a K0.5 of 0. 8 microM and a Hill coefficient of 1.0. Neither of the identified Ca2+ binding regions contained an EF-hand motif. We conclude that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, which are localized in different structural regions of the protein.

Amino Acid Sequence↗
PubMed1996

Purkinje neurons express the SERCA3 isoform of the organellar type Ca(2+)-transport ATPase.

We report the distribution of the sarco(endo)plasmic reticulum Ca2+ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca2+ pump has an approximately 5-fold lower affinity for Ca2+ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca(2+)-pump isoform could have important functional implications for the regulation of the cytosolic Ca2+ concentration in Purkinje neurons.

Animals↗
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

Expression and function of ryanodine receptors in nonexcitable cells.

We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses.

Animals↗
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↗