Search PubMed⌕ Search

Biomedical subjects

M J Berridge

Publications and source records attributed to M J Berridge.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of elementary Ca2+ release signals in NGF-differentiated PC12 cells and hippocampal neurons.

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.

Animals↗
PubMed1999

Elementary [Ca2+]i signals generated by electroporation functionally mimic those evoked by hormonal stimulation.

The generation of oscillations and global Ca2+ waves relies on the spatio-temporal recruitment of elementary Ca2+ signals, such as 'Ca2+ puffs'. Each elementary signal contributes a small amount of Ca2+ into the cytoplasm, progressively promoting neighboring Ca2+ release sites into an excitable state. Previous studies have indicated that increases in frequency or amplitude of such hormone-evoked elementary Ca2+ signals are necessary to initiate Ca2+ wave propagation. In the present study, an electroporation device was used to rapidly and reversibly permeabilize the plasma membrane of HeLa cells and to allow a limited influx of Ca2+. With low field intensities (100-500 V/cm), brief (50-100 micros) electroporation triggered localized Ca2+ signals that resembled hormone-evoked Ca2+ puffs, but not global signals. With such low intensity electroporative pulses, the Ca2+ influx component was usually undetectable, confirming that the electroporation-induced local signals represented Ca2+ puffs arising from the opening of intracellular Ca2+ release channels. Increasing either the frequency at which low-intensity electroporative pulses were applied, or the intensity of a single electroporative pulse (>500 V/cm), resulted in caffeine-sensitive regenerative Ca2+ waves. We suggest that Ca2+ puffs caused by electroporation functionally mimic hormone-evoked elementary events and can activate global Ca2+ signals if they provide a sufficient trigger.

Calcium↗
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↗
PubMed1997

The 1996 Massry Prize. Inositol trisphosphate and calcium: two interacting second messengers.

Inositol trisphosphate (InsP3) functions as a second messenger to control the release of internal calcium and the entry of external calcium. This InsP3/Ca2+ signalling pathway is based on a hierachical system with the release from individual channels being the fundamental event (Ca2+ blips). A collection of blips produces larger elementary events (Ca2+ puffs) which then fuse through a regenerative process of calcium-induced calcium release to give global events (Ca2+ waves). This highly versatile signalling system has been adapted to regulate a wide range of cellular processes including fertilization, development, proliferation and apoptosis.

Animals↗
PubMed1997

Elementary and global aspects of calcium signalling.

Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.

Animals↗
PubMed1997

Lymphocyte activation in health and disease.

Lymphocytes employ a complex assembly of signaling elements that have been organized on a spatiotemporal map to define their role in stimulating both proliferation and apoptosis. The antigen/major histocompatibility complex (MHC) initiates the sequence by organizing the assembly of an active T-cell receptor (TCR) complex responsible for transmitting information down various signaling cassettes (e.g., the IP3/Ca2+, DAG/PKC, ras/MAPK, and the PI 3-K pathways). It is proposed that CD28 may exert its costimulatory action by facilitating the assembly of an effective scaffold of signaling elements within the TCR complex. The absence of costimulation through CD28 seems to result in the assembly of a defective scaffold that reverses slowly and may thus account for the state of unresponsiveness responsible for peripheral T-cell tolerance. The signaling cassettes activated by the TCR and CD28 then engage cytosolic factors that transmit information into the nucleus to activate the genes that code for the IL-2 and Fas signaling pathways. The IL-2 and Fas receptors employ additional signaling cassettes (e.g., the JAK/STAT and the sphingomyelinase/ceramide pathways) to mediate their effects on proliferation and apoptosis, respectively. Information concerning these signaling systems is beginning to provide therapeutic strategies to manipulate the immune system to overcome human immunodeficiency virus (HIV) infection, autoimmune diseases, and graft rejection.

Animals↗
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

Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ spiking.

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLC beta 1, PLC beta 3 and PLC gamma isoenzymes. Furthermore, immunodetection of PLC gamma 1 using monoclonal antibodies reveals that PLC gamma 1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLC beta protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 microM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 microM), herbimycin (10 microM) and geldanamycin (0.6 microM) which could affect signalling through PLC gamma hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.

Acetylcholine↗
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↗
PubMed1996

Capacitative calcium entry is colocalised with calcium release in Xenopus oocytes: evidence against a highly diffusible calcium influx factor.

Depletion of intracellular calcium stores activates the plasma membrane capacitative calcium entry pathway in many cell types. The nature of the signal that couples the depletion of the intracellular calcium stores to the activation of the plasma membrane calcium influx pathway is as yet unknown. It has recently been suggested that a highly diffusible calcium influx factor is involved in the activation of capacitative calcium entry, and that its action is potentiated by the protein phosphatase inhibitor okadaic acid. Depletion of intracellular calcium stores in a localised region of a Xenopus oocyte was found to evoke capacitative calcium entry exclusively colocalised across the stimulated area of the plasma membrane, arguing against the involvement of a highly diffusible calcium influx factor. Equally, no evidence could be found for the presence of a soluble calcium influx factor in the bulk cytosol of Xenopus oocytes. The potentiation of capacitative calcium entry by okadaic acid resembled that mediated by the activation of protein kinase C, thus suggesting that okadaic acid activity may not necessarily be related to the action of a putative calcium influx factor.

Animals↗