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M D Bootman

Publications and source records attributed to M D Bootman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Hormone-evoked elementary Ca2+ signals are not stereotypic, but reflect activation of different size channel clusters and variable recruitment of channels within a cluster.

Previous studies of (InsP3)-evoked elementary Ca2+ events suggested a hierarchy of signals; fundamental events ("Ca2+ blips") arising from single InsP3 receptors (InsP3Rs), and intermediate events ("Ca2+ puffs") reflecting the coordinated opening of a cluster of InsP3Rs. The characteristics of such elementary Ca2+ release signals provide insights into the functional interaction and distribution of InsP3Rs in living cells. Therefore we investigated whether elementary Ca2+ signaling is truly represented by such stereotypic release events. A histogram of >900 events revealed a wide spread of signal amplitudes (20-600 nM; mean 216 +/- 4 nM; n = 206 cells), which cannot be explained by stochastic variation of a stereotypic Ca2+ release site. We identified elementary Ca2+ release sites with consistent amplitudes (<20% difference) and locations with variable amplitudes (approximately 500% difference). Importantly, within single cells, distinct sites displayed events with significantly different mean amplitudes. Additional determinants affecting the magnitude of elementary Ca2+ release were identified to be (i) hormone concentration, (ii) day-to-day variability, and (iii) a progressively decreasing Ca2+ release during prolonged stimulation. We therefore suggest that elementary Ca2+ events are not stereotypic, instead a continuum of signals can be achieved by either recruitment of entire clusters with different numbers of InsP3Rs or by a graded recruitment of InsP3Rs within a cluster.

Calcium↗
PubMed1998

Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors.

Capacitative Ca2+ entry (CCE) is a universal mechanism for refilling intracellular Ca2+ stores in electrically non-excitable cells. The situation in excitable cells is less clear, however, since they may rely on other entry mechanisms for Ca2+-store refilling. In the present study we investigated CCE in intact PC12 cells, using acetylcholine to bring about activation of InsP3 receptors (InsP3Rs), caffeine to activate ryanodine receptors (RyRs) and thapsigargin to inhibit sarco/endoplasmic reticulum Ca2+-ATPase pumps. We found that depletion of the InsP3-, caffeine- or thapsigargin-sensitive stores promoted Ca2+ entry, suggesting that stimulation of either InsP3Rs or RyRs can activate CCE. The CCE pathways activated by InsP3Rs, RyRs and thapsigargin appeared to be independent at least in part, since their effects were found to be additive. However, CCE triggered by caffeine, acetylcholine or thapsigargin progressively diminished with time. The decay of CCE caused by one agent also inhibited subsequent responses to the others, suggesting that some component of the CCE pathway is common to all intracellular Ca2+ stores. The magnitude of CCE stimulated by InsP3Rs or RyRs was related to the size of the stores; the InsP3-sensitive store was smaller than the RyR-sensitive store and triggered a smaller entry component. However, both stores filled with a similar half time (about 1 min), and both could be filled more rapidly by depolarization-induced Ca2+ entry through voltage-operated channels. A significant basal Ca2+ influx was apparent in PC12 cells. The basal entry component may be under the control of the InsP3-sensitive Ca2+ store, since short incubations in Ca2+-free medium depleted this store.

Acetylcholine↗
PubMed1997

Nuclear calcium signalling by individual cytoplasmic calcium puffs.

It is known that the nucleoplasmic ionised calcium concentration (Can) controls nuclear functions such as transcription, although the source and nature of the signals which modulate Can are unclear. Using confocal imaging, we investigated the subcellular origin of Can signals in Fluo-3-loaded HeLa cells. Our data indicate that all signals which increased Can were of cytoplasmic origin. Can was elevated during the propagation of global Ca waves within cells. More strikingly, we found that individual cytoplasmic elementary release events e.g. Ca puffs, evoked by physiological levels of stimulation, caused transient Can increases. Significantly, >70% of all Ca puffs originated within a 2-3 micron perinuclear zone and propagated anisotropically across the entire nucleus. Due to the relatively slow relaxation of Can transients compared with those in the cytoplasm, repetitive perinuclear Ca puffs were integrated into a 'staircase' of increasing Can. Due to the effective diffusion of Ca in the nucleoplasm, the nucleus served as a 'Ca tunnel', distributing Ca to parts of the cytosol which were otherwise not within the cytoplasmic diffusion radii of Ca puffs. Given the close proximity of the majority of puff sites to the nucleus, it seems that the elementary Ca release system is designed to facilitate nuclear Ca signalling. Consequently, Ca-dependent regulation of nuclear function must be considered at the microscopic elementary level.

Biological Transport↗
PubMed1997

Cooking with calcium: the recipes for composing global signals from elementary events.

Recent studies have suggested that global intracellular Ca2+ signals arise from the summation and coordination of subcellular elementary release events (e.g., "Ca2+ puffs"), although the modes of recruitment of such signals are unknown. In order to understand how cells utilize elementary Ca2+ release events, we imaged Ca2+ transients evoked through the phosphoinositide pathway in HeLa cells using confocal microscopy. During the pacemaker phase leading to the global Ca2+ signal, elementary Ca2+ release events were recruited in (1) frequency, (2) amplitude, and (3) spatial domains. Since each digital elementary event contributes to a small change of the analog cytosolic Ca2+ concentration, the net effect of the advancement in the three domains is to drive the ambient Ca2+ concentration toward a threshold where the signal becomes regenerative, resulting in a global Ca2+ wave.

Calcium↗
PubMed1996

Subcellular Ca2+ signals underlying waves and graded responses in HeLa cells.

BACKGROUND: Many agonist-evoked intracellular Ca2+ signals have a complex spatio-temporal arrangement, and are observed as repetitive Ca2+ spikes and Ca2+ waves. The key to revealing how these complex signals are generated lies in understanding the functional structure of the intracellular Ca2+ pool. Previous imaging studies, using relatively large cells such as oocytes and myocytes, have identified subcellular elementary Ca2+ signals, indicating that the intracellular Ca2+ pool releases Ca2+ from functionally discrete sites. However, it is unclear whether the intracellular Ca2+ pool in smaller cells has a similar architecture, and how such subcellular signals would contribute to global spikes and waves. RESULTS: We detected subcellular Ca2+ signals during the response of single Fura2-loaded HeLa cells to histamine. The spatio-temporal properties of some of these signals were similar to the elementary Ca2+ signals observed in other cells. Subcellular Ca2+ signals were particularly obvious during the 'pacemaker' Ca2+ rise that preceded the regenerative Ca2+ wave. During this pacemaker, the Ca2+ signals were observed initially in the region from which the Ca2+ wave originated, but became more widespread and frequent until a Ca2+ wave was spawned. Similar localized signals were seen during the post-wave Ca2+ increase, and during the low-amplitude Ca2+ responses evoked by threshold histamine concentrations. CONCLUSIONS: The intracellular Ca2+ pool in HeLa cells is composed of many functionally discrete units. Upon stimulation, these units produce localized Ca2+ signals. The sequential activation and summation of these units results in Ca2+ wave propagation and, furthermore, the differential recruitment of these units may underlie the graded amplitude of the intracellular Ca2+ signals.

Animals↗
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

Extracellular calcium concentration controls the frequency of intracellular calcium spiking independently of inositol 1,4,5-trisphosphate production in HeLa cells.

Stimulation of single HeLa cells with histamine evoked repetitive increases of the intracellular calcium ion concentration (Ca2+ spikes). The frequency of Ca2+ spiking increased as the extracellular hormone concentration was elevated. In addition, the frequency of Ca2+ spiking could be accelerated by increasing the extracellular Ca2+ concentration ([Ca2+]0) in the presence of a constant hormone concentration. The range of [Ca2+]0 over which the spiking frequency could be titrated was nominally-zero to 10mM, being half-maximally effective at approx. 1 and 2.5mM for 37 and 22 degrees C respectively. The effect of [Ca2+]0 on inositol phosphates production was also examined. Changes of [Ca2+]0 over a range which had been found to affect the frequency of Ca2+ spiking did not have any effect on the rate of myo-inositol 1,4,5-trisphosphate (InsP3) production, although an increase in inositol phosphates production was observed as [Ca2+]0 was increased from zero to values giving less than half-maximal Ca2+ spike frequency. These data suggest that at low Ca2+ spike frequency, Ca2+-stimulated activation of phospholipase C may contribute to Ca2+ spiking in HeLa cells, but under some conditions the availability of Ca2+ to the intracellular stores, rather than changes in the rate of InsP3 production, determines the Ca2+ spike frequency.

Calcium↗
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

Smoothly graded Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores.

Stimulation of cells with Ca(2+)-mobilizing hormones often leads to the generation of temporally and spatially complex changes in the intracellular Ca2+ ion concentration ([Ca2+]i). To understand the mechanisms regulating Ca2+ release from intracellular stores more clearly, we investigated the ability of histamine to release Ca2+ stores under different experimental conditions, using video imaging of single Fura-2-loaded HeLa cells. In Ca(2+)-free medium, stepwise increases in histamine concentration released an increasing proportion of the intracellular Ca2+ pool. This pattern of Ca2+ release is analogous to the "quantal" release of Ca2+ previously observed using permeabilized cells. Quantal Ca2+ release was observed at both 20 and 37 degrees C and was not due to inactivation or desensitization of the Ca2+ release mechanism, since application of histamine in a pulsatile manner, which avoided desensitization of the Ca(2+)-release mechanism, still produced a quantal response. In Ca(2+)-containing medium at both 20 and 37 degrees C, stepwise increases in histamine concentration evoked [Ca2+]i responses where the amplitude was smoothly graded in direct proportion to the histamine concentration. Similar smoothly graded responses were observed from HeLa cells in Ca(2+)-free medium. These data indicate that hormone-evoked Ca2+ release from intracellular stores is limited by the hormone concentration, and that the mechanisms underlying complex [Ca2+]i signals do not lead to an all-or-none release of Ca2+ from the entire intracellular Ca2+ pool. We suggest that the hormone-sensitive intracellular Ca2+ pool is composed of functionally discrete units that are recruited by agonists in a concentration-dependent manner.

Calcium↗
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↗