Is there a role of C-reactive protein in red blood cell aggregation?

INTRODUCTION: Numerous clinical studies related the plasma level of C-reactive protein (CRP) to the erythrocyte sedimentation rate (ESR) independent of the kind of disease. The molecular regulation of the process is unknown. METHODS: We performed a meta-analysis of 10 previous studies and experimentally probed for a direct action of CRP on red blood cells (RBCs) by different methods including determination of a microscopic aggregation index, Ca(2+) imaging and analysis of sedimentation experiments. RESULTS: The meta-analysis revealed a statistically significant correlation (Pearson coefficient of 0.37; P < 0.0001), but we could not find any experimental evidence for a direct CRP-RBC interaction. Instead, we could confirm a correlation between fibrinogen level and ESR. CONCLUSION: Therefore, we concluded that CRP and ESR cannot account for nor replace each other as a diagnostic measure. The correlation between CRP level and ESR is most probably caused by fibrinogen, because its increase coincides with elevated CRP levels.

Study on the removal efficiency of UF membranes using bacteriophages in bench-scale and semi-technical scale.

To determine the removal efficiency of ultrafiltration (UF) membranes for nano-particles in the size range of viruses the state of the art uses challenge tests with virus-spiked water. This work focuses on bench-scale and semi-technical scale experiments. Different experimental parameters influencing the removal efficiency of the tested UF membrane modules were analyzed and evaluated for bench- and semi-technical scale experiments. Organic matter in the water matrix highly influenced the removal of the tested bacteriophages MS2 and phiX174. Less membrane fouling (low ΔTMP) led to a reduced phage reduction. Increased flux positively affected phage removal in natural waters. The tested bacteriophages MS2 and phiX174 revealed different removal properties. MS2, which is widely used as a model organism to determine virus removal efficiencies of membranes, mostly showed a better removal than phiX174 for the natural water qualities tested. It seems that MS2 is possibly a less conservative surrogate for human enteric virus removal than phiX174. In bench-scale experiments log removal values (LRV) for MS2 of 2.5-6.0 and of 2.5-4.5 for phiX174 were obtained for the examined range of parameters. Phage removal obtained with differently fabricated semi-technical modules was quite variable for comparable parameter settings, indicating that module fabrication can lead to differing results. Potting temperature and module size were identified as influencing factors. In conclusion, careful attention has to be paid to the choice of experimental settings and module potting when using bench-scale or semi-technical scale experiments for UF membrane challenge tests.

Consideration of emerging pollutants in groundwater-based reuse concepts.

Elimination of pathogens and emerging pollutants represents a key factor in integrated water resources management in arid regions. Within the SMART Jordan Valley project it is the objective of this study to assess the occurrence and examine the elimination of selected emerging pollutants and pathogens in waste water treatment and aquifer recharge. In batch and soil column studies non-chlorinated organophosphorous compounds (tri-n-butylphosphate, triphenylphosphate) and endocrine disruptors (e.g. 17-ß-estradiol, bisphenol A) proved to be biodegradable, while the X-ray contrast agents iomeprol and iopromide were eliminated in the soil columns only, and the chlorinated trialkylphosphates showed persistency. Treating waste water in a membrane bioreactor (MBR) in combination with powdered activated carbon (PAC) resulted in considerable removal rates also for the more persistent compounds such as the antiepileptic carbamazepine. Viruses were shown to be present in most of the Jordan Valley surface water samples. MBR treatment resulted in a decrease of MS2 bacteriophages used as model viruses.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

Calcium puffs are generic InsP(3)-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses.

Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.

FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments.

FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

The organisation and functions of local Ca(2+) signals.

Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca(2+) to play a pivotal role in cell biology results from the facility that cells have to shape Ca(2+) signals in space, time and amplitude. To generate and interpret the variety of observed Ca(2+) signals, different cell types employ components selected from a Ca(2+) signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca(2+) signals that suit their physiology. Recent studies have demonstrated the importance of local Ca(2+) signals in defining the specificity of the interaction of Ca(2+) with its targets. Furthermore, local Ca(2+) signals are the triggers and building blocks for larger global signals that propagate throughout cells.

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals.

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.

Predetermined recruitment of calcium release sites underlies excitation-contraction coupling in rat atrial myocytes.

Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37 degrees C using rapid confocal microscopy. Within a few milliseconds of electrical excitation, spatially discrete subsarcolemmal Ca2+ signals were initiated. Twenty to forty milliseconds after stimulation the spatial overlap of these Ca2+ signals gave a 'ring' of elevated Ca2+ around the periphery of the cells. However, this ring was not continuous and substantial Ca2+ gradients were observed. The discrete subsarcolemmal Ca2+-release sites, which responded in a reproducible sequence to repetitive depolarisations and displayed the highest frequencies of spontaneous Ca2+ sparks in resting cells, were denoted 'eager sites'. Immunostaining atrial myocytes for type II ryanodine receptors (RyRs) revealed both subsarcolemmal 'junctional' RyRs, and also 'non-junctional' RyRs in the central bulk of the cells. A subset of the junctional RyRs comprises the eager sites. For cells paced in the presence of 1 mM extracellular Ca2+, the response was largely restricted to a subsarcolemmal 'ring', while the central bulk of the cell displayed a approximately 5-fold lower Ca2+ signal. Under these conditions the non-junctional RyRs were only weakly activated during E-C coupling. However, these channels are functional and the Ca2+ stores were at least partially loaded, since substantial homogeneous Ca2+ signals could be stimulated in the central regions of atrial myocytes by application of 2.5 mM caffeine. Neither the location nor activation order of the eager sites was affected by increasing the trigger Ca2+ current (by increasing extracellular Ca2+ to 10 mM) or the sarcoplasmic reticulum (SR) Ca2+ load (following 1 min incubation in 10 mM extracellular Ca2+), although with increased SR Ca2+ load, but not greater Ca2+ influx, the delay between the sequential activation of eager sites was reduced. In addition, increasing the trigger Ca2+ current or the SR Ca2+ load changed the spatial pattern of the Ca2+ response, in that the Ca2+ signal propagated more reliably from the subsarcolemmal initiation sites into the centre of the cell. Due to the greater spatial spread of the Ca2+ signals, the averaged global Ca2+ transients increased by approximately 500 %. We conclude that rat atrial myocytes display a predetermined spatiotemporal pattern of Ca2+ signalling during early E-C coupling. A consistent set of eager Ca2+ release sites with a fixed location and activation order on the junctional SR serve to initiate the cellular response. The short latency for activation of these eager sites suggests that they reflect clusters of RyRs closely coupled to voltage-operated Ca2+ channels in the sarcolemma. Furthermore, their propensity to show spontaneous Ca2+ sparks is consistent with an intrinsically higher sensitivity to Ca2+-induced Ca2+ release. While the subsarcolemmal Ca2+ response can be considered as stereotypic, the central bulk of the cell grades its response in direct proportion to cellular Ca2+ load and Ca2+ influx.

Calcium signalling--an overview.

Calcium (Ca2+) is an almost universal intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in the dimensions of space, time and amplitude. To generate the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling 'toolkit', which comprizes an array of signalling, homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology.

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation.

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.

Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.

Calcium-modulating cyclophilin ligand desensitizes hormone-evoked calcium release.

The Ca(2+)-modulating cyclophilin ligand (CAML) protein causes stimulation of transcription factors via activation of a store-operated Ca(2+) entry pathway. Since CAML is widely expressed in mammalian tissues, it may be an important regulator of Ca(2+) store function. In the present study, we investigated the consequence of CAML overexpression on Ca(2+) signaling using rapid confocal imaging of Fluo3-loaded NIH3T3 fibroblasts. Control and CAML-expressing cells gave concentration-dependent responses to the Ca(2+) mobilizing agonist ATP. CAML expression reduced the sensitivity of the cells so that higher concentrations of ATP were needed to achieve global Ca(2+) waves. The amplitudes of Ca(2+) waves were significantly reduced in CAML expressing cells, consistent with earlier suggestions that CAML causes depletion of internal Ca(2+) stores. With low ATP concentrations, only local Ca(2+) release events were observed. CAML did not affect the characteristics of these local Ca(2+) signals, suggesting that it does not directly affect Ca(2+) release channels.

Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production. Potential involvement of endogenous EDG-4 receptors.

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.

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.

Nuclear calcium signalling.

The topic of nuclear Ca2+ signalling is beset by discrepant observations of substantial nuclear/cytoplasmic gradients. The reasons why some labs have recorded such gradients, whilst other workers see equilibration of Ca2+(cyt) and Ca2+(nuc) using the same cells and techniques, is unexplained. Furthermore, how such gradients could arise across the NE that possesses many highly-conductive NPCs is a mystery. Although nuclei may have the capacity to be autonomous signalling entities, with functional Ca2+ release channels and an inositide cycle, the balance of evidence suggests that Ca2+ release on the inner NE does not occur during physiological stimulation. Our work suggests that elementary Ca2+ release events originating in the cytoplasm can give rise to Ca2+ signals without causing elevation of the bulk cytoplasm. Clearly, the many Ca2+ signalling mechanisms that may impinge on Ca2+(nuc) will remain a topic of controversy and debate for some time.

Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization.

We investigated the consequences of depolarizing the mitochondrial membrane potential (Deltapsi(mit)) on Ca(2+) signals arising via inositol 1,4,5-trisphosphate receptors (InsP(3)R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize Deltapsi(mit). Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca(2+)-mobilizing hormone and prolonged the recovery of cytosolic Ca(2+) after washout of the hormone; effects consistent with the removal of a passive Ca(2+) buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca(2+) changes were observed to progressively decline. The inhibition of Ca(2+) signalling was observed using different Ca(2+)-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P(3) ester. Upon washout of FCCP, the Ca(2+) signals recovered with a time course similar to the re-establishment of Deltapsi(mit). Global measurements indicated that none of the obvious factors such as changes in pH, ATP concentration, cellular redox state, permeability transition pore activation or reduction in Ca(2+)-store loading appeared to underlie the inhibition of Ca(2+) signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing Deltapsi(mit), prevents InsP(3)R activation.

Microscopic properties of elementary Ca2+ release sites in non-excitable cells.

BACKGROUND: Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1 ,4,5,-trisphosphate (InsP3) receptors are the building blocks for local and global Ca2+ signalling. We previously found that one, or a few, Ca2+ puff sites within agonist-stimulated cells act as 'pacemakers' to initiate global Ca2+ waves. The factors that distinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown. RESULTS: The spatiotemporal properties of Ca2+ puffs were investigated using confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ puff sites were activated during stimulation of cells with different agonists. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskeleton. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP3 receptors were directly stimulated with thimerosal or membrane-permeant InsP3 esters. Immunostaining indicated that the perinuclear position of pacemaker Ca2+ puffs was not due to the localised expression of InsP3 receptors. CONCLUSIONS: The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP3 sensitivity.