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M D Bootman

Publications and source records attributed to M D Bootman.

49 records · Page 3Linked to original sources
PubMed1994

Quantal Ca2+ mobilization by ryanodine receptors is due to all-or-none release from functionally discrete intracellular stores.

Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.

Adrenal Medulla↗
PubMed1994

Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells.

Thrombin and the thrombin receptor agonist peptide (TRAP) caused a rise in intracellular calcium concentration ([Ca2+]i) in the human osteoblast-like cell line Saos-2. Striking differences in the [Ca2+]i signals elicited by these agonists were revealed. In cell populations, thrombin induced a transient increase in [Ca2+]i while TRAP caused a biphasic [Ca2+]i response consisting of an initial peak and a sustained plateau phase. In individual cells, thrombin mainly caused a single [Ca2+]i transient while TRAP induced repetitive [Ca2+]i spikes. Neither tyrosine phosphorylation, cAMP-dependent phosphorylation, nor pertussis toxin-sensitive G proteins appeared to be involved in thrombin receptor [Ca2+]i signaling in this cell line. However, the sustained [Ca2+]i response caused by TRAP was converted into a transient, thrombin-like response by pretreatment with serine/threonine phosphatase inhibitors. Pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) abrogated thrombin receptor [Ca2+]i signaling, and TRAP-induced Ca2+ entry was inhibited by the acute treatment with PMA. In contrast, Ca2+ entry stimulated by thapsigargin was not sensitive to agents affecting serine/threonine phosphorylation. The observation that thrombin and TRAP, despite being agonists for a common receptor, induce dissimilar [Ca2+]i responses indicates that binding of TRAP alone is insufficient to fully regulate the thrombin receptor in Saos-2 cells.

Amino Acid Sequence↗
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↗
PubMed1993

Quantal Ca2+ release from caffeine-sensitive stores in adrenal chromaffin cells.

In populations of fura-2-loaded chromaffin cells, caffeine caused a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), in the presence or absence of external Ca2+ ([Ca2+]o), that was saturable, reversible, and inhibited in a use-dependent fashion by ryanodine. These data confirm that caffeine mobilizes Ca2+ from the ryanodine-sensitive intracellular stores in chromaffin cells. In nominally Ca(2+)-free medium, sustained stimulation of cell populations or single cells with low caffeine concentrations failed to completely empty the caffeine-sensitive stores. In each case, there was a transient [Ca2+]i elevation, but a subsequent challenge with a higher caffeine concentration evoked a further [Ca2+]i rise, indicating that Ca2+ stores within individual cells were heterogeneous in their sensitivities to caffeine and that caffeine-induced Ca2+ release was quantal. The heterogeneous sensitivity was also demonstrated using ryanodine; pretreatment of cell populations with increasing caffeine concentrations with a constant ryanodine concentration, caused a dose-dependent irreversible inhibition of the response to the subsequent addition of a maximal caffeine concentration. We conclude that, within single chromaffin cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to caffeine and the fraction of Ca2+ stores mobilized by caffeine increases in direct proportion to the caffeine concentration.

Adrenal Medulla↗
PubMed1993

Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells.

The receptor responsible for the increase in intracellular calcium concentration ([Ca2+]i) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was observed with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin receptor is activated by thrombin cleavage to create a new NH2 terminus that acts as a tethered ligand, and peptides based on the tethered ligand can activate the receptor independently of thrombin. Northern analysis indicated the presence of mRNA encoding the platelet receptor in Saos-2 cells, and surface expression of the receptor was demonstrated by immunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single-letter amino acid code) was found to increase [Ca2+]i. The maximal response to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 +/- 6 microM peptide. The time course of the increase in [Ca2+]i with the peptide was different than that observed with thrombin; a pronounced shoulder was observed after an initial sharp rise. The phenylalanine in the second position of the agonist peptide and the arginine in the fifth position were shown to be essential for its activity. The requirement for proteolysis of the receptor for the thrombin-dependent increase in [Ca2+]i was demonstrated by two methods. Antibodies that reacted with the cleavage site of the receptor abolished the effect of thrombin on [Ca2+]i. In addition, a mutant of thrombin without catalytic activity as well as chemically inactivated thrombin failed to cause an increase in [Ca2+]i. Similar results were obtained with the rat osteoblast-like cell line UMR-106; a tethered ligand peptide based on the rat sequence induced an increase in [Ca2+]i, and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.

Amino Acid Sequence↗
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

Calcium regulation in tissue-cultured human and bovine lens epithelial cells.

PURPOSE: To study calcium regulatory mechanisms in lens cells with particular reference to the relative contributions from the calcium adenosine triphosphatase of plasma and endoplasmic reticulum membranes, respectively. METHODS: The calcium-sensitive fluorescent dye, Fura 2, was incorporated into tissue-cultured human and bovine epithelial cells and internal calcium was calibrated using the ionomycin (1 microM) method. The dynamics of calcium release from the endoplasmic reticulum were also studied in digitonin-permeabilized bovine cells. RESULTS: Tissue-cultured bovine and human lens cells have very similar resting calcium levels (235 +/- 22 nM and 216 +/- 12 nM, respectively). Thapsigargin caused an increase in cytoplasmic calcium both in the presence and absence of external calcium, but the calmodulin antagonist W7 only initiated an increase in the presence of external Ca2+. The effects of thapsigargin and W7 were additive. Exposing lens cells to Na(+)-free perfusing solutions caused a transient increase in internal Ca2+. Bovine lens cells permeabilized by digitonin-released Ca2+ when exposed to inositol (1,4,5) triphosphate and the effect was maximal at 1 microM. CONCLUSIONS: Lens cytoplasmic calcium is controlled by calcium adenosine triphosphatases at the plasma and endoplasmic reticulum membranes. The former is inhibited by W7 and insensitive to thapsigargin whereas the latter is inhibited by thapsigargin, but insensitive to W7. The lens endoplasmic reticulum store is also controlled by an inositol (1,4,5) trisphosphate calcium-release mechanism. Na+/Ca2+ exchange plays a relatively minor role in calcium regulation, at least at resting calcium levels.

Aged↗
PubMed1992

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.

Calcium↗
PubMed1992

All-or-nothing Ca2+ mobilization from the intracellular stores of single histamine-stimulated HeLa cells.

1. Histamine-stimulated mobilization of intracellular Ca2+ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca2+ indicator Fura-2 and 45Ca2+ measurements. Digital video imaging of Fura-2-loaded cells was used to measure the intracellular calcium concentration ([Ca2+]i) of single cells. 2. In populations of HeLa cells, histamine caused a concentration-dependent increase in cytoplasmic [Ca2+]. The initial transient increase was independent of extracellular Ca2+ (Ca2+o) and was followed by a sustained increase that was abolished by removal of Ca2+o. 3. In Ca(2+)-free medium ([Ca2+]o < 1 microM), a maximal histamine concentration (25 microM) caused a transient increase in [Ca2+]i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores had been completely emptied. Lower concentrations of histamine (0.5-10 microM) caused smaller, concentration-dependent increases in [Ca2+]i that were also transient. After exposure to these low histamine concentrations, where [Ca2+]i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca2+]i. The second increase in [Ca2+]i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca2+ released in the initial response was not substantially resequestered into histamine-sensitive stores. 4. Single HeLa cells challenged with low concentrations of histamine in Ca(2+)-free medium responded with transient increases in [Ca2+]i, but individual cells differed in their sensitivity with 51% of cells responding to 1 microM, and 98% responding to 25 microM-histamine. 5. When single cells in Ca(2+)-free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca2+]i increase after which [Ca2+]i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca2+]i increase evoked by a subsequent histamine addition. 6. Unidirectional 45Ca2+ efflux from saponin-permeabilized HeLa cells showed that, under conditions that prevented Ca2+ resequestration, submaximal concentrations of InsP3 rapidly emptied only a fraction of the InsP3-sensitive Ca2+ stores. The failure of low InsP3 concentrations to fully mobilize the InsP3-sensitive Ca2+ stores was not a consequence of InsP3 degradation. 7. We conclude that within single HeLa cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to InsP3, and the fraction of Ca2+ stores mobilized by InsP3 increases as the InsP3 concentration increases.

Biological Transport↗
PubMed1990

Two sulphonated dye compounds which compete for inositol 1,4, 5-trisphosphate binding to rat liver microsomes: effects on 5'-phosphatase activity.

The ability of heparin to interact with the Ins 1,4,5-P3 receptor is dependent on its chain length and degree of sulphation. Here we report results obtained with two sulphonated dye compounds of known structures and molecular weights below 1000, cibacron blue and Patent blue. Both compounds compete for Ins 1,4,5-P3 binding to rat liver microsomes and also inhibit Ins 1,4,5-P3 5'-phosphatase activity in the same preparation. Comparison with the effects of heparin show these to be two separate actions of the compounds.

Animals↗
PubMed1989

The effect of heparin on the inositol 1,4,5-trisphosphate receptor in rat liver microsomes. Dependence on sulphate content and chain length.

Heparin is known to inhibit the binding of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) to high-affinity binding sites and to inhibit Ins 1,4,5-P3-induced Ca2+ release from intracellular membrane-bound stores [(1987) J. Biol. Chem. 262, 12132-12136; (1987) FEBS Lett. 228, 57-59]. We have performed studies to clarify the structural requirements for this action of heparin in rat liver microsomes. Both N- and O-linked sulphate groups contribute to binding activity, since de-N-sulphated heparin was without effect on the Ins 1,4,5-P3 receptor whereas a polyxylan bearing only O-linked sulphates (pentosan polysulphate) was as active as heparin. Therefore, the density of negative charge contributed by sulphate groups is important for the binding of heparin. Heparins with high and low affinity for antithrombin III both inhibited Ins 1,4,5-P3 binding. There was a strong dependence on chain length, since binding activity decreased dramatically as the size of the heparin chain was reduced below that of 18-24 monosaccharide units.

Animals↗
PubMedDate not supplied

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart.

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.

Animals↗