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The field of quantum computing has grown from concept to demonstration devices over the past 20 years. Universal quantum computing offers efficiency in approaching problems of scientific and commercial interest, such as factoring large numbers, searching databases, simulating intractable models from quantum physics, and optimizing complex cost functions. Here, we present an 11-qubit fully-connected, programmable quantum computer in a trapped ion system composed of 13 171Yb+ ions. We demonstrate average single-qubit gate fidelities of 99.5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}%, average two-qubit-gate fidelities of 97.5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}%, and SPAM errors of 0.7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}%. To illustrate the capabilities of this universal platform and provide a basis for comparison with similarly-sized devices, we compile the Bernstein-Vazirani and Hidden Shift algorithms into our native gates and execute them on the hardware with average success rates of 78\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}% and 35\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}%, respectively. These algorithms serve as excellent benchmarks for any type of quantum hardware, and show that our system outperforms all other currently available hardware., The growing complexity of quantum computing devices makes presents challenges for benchmarking their performance as previous, exhaustive approaches become infeasible. Here the authors characterise the quality of their 11-qubit device by successfully computing two quantum algorithms. more...

Human platelets regulate agonist-evoked Ca2+ signalling through Ca2+ release from and sequestration into acidic organelles. Previous studies have pharmacologically characterised the presence of a Ca2+-H+ exchanger in these organelles. This exchanger appears to regulate a secondary plateau phase in agonist-evoked cytosolic Ca2+ signals in fura-2-loaded human platelets. Here we demonstrate that cytochalasin D treatment removes the secondary plateau in ADP-evoked Ca2+ signals elicited in the absence of external Ca2+. This effect was reversed by pretreatment with nigericin, a K+/H+ exchanger that short-circuits the Ca2+-H+ exchanger. Using Fluo-5N- or Lysosensor Green-loaded cells, cytochalasin D was found to enhance Ca2+ sequestration into acidic organelles by preventing their alkalinisation. Additional experiments demonstrated that ADP-evoked alkalinisation of acidic organelles and subsequent slowing of acidic organellar Ca2+ sequestration was mediated by autocrine 5-HT signalling. Enhancing this 5-HT signalling using fluoxetine overcame the inhibitory effect of cytochalasin D on ADP-evoked Ca2+ signals, indicating that cytochalasin D interferes with 5-HT autocrine signalling. The ability of Cytochalasin D to interfere with autocrine 5-HT signalling was downstream of the 5-HT2A receptor as secretion of [3H]-5-HT from ADP-stimulated human platelets was not reduced. These data provide the first evidence that the pH gradient across acidic organelles is dynamically regulated upon human platelet activation, and that this can play a significant role in controlling human platelet function by modulating Ca2+-H+ exchange and so [Ca2+]i. more...

Quantum computing leverages the quantum resources of superposition and entanglement to efficiently solve computational problems considered intractable for classical computers. Examples include calculating molecular and nuclear structure, simulating strongly interacting electron systems, and modeling aspects of material function. While substantial theoretical advances have been made in mapping these problems to quantum algorithms, there remains a large gap between the resource requirements for solving such problems and the capabilities of currently available quantum hardware. Bridging this gap will require a co-design approach, where the expression of algorithms is developed in conjunction with the hardware itself to optimize execution. Here we describe an extensible co-design framework for solving chemistry problems on a trapped-ion quantum computer and apply it to estimating the ground-state energy of the water molecule using the variational quantum eigensolver (VQE) method. The controllability of the trapped-ion quantum computer enables robust energy estimates using the prepared VQE ansatz states. The systematic and statistical errors are comparable to the chemical accuracy, which is the target threshold necessary for predicting the rates of chemical reaction dynamics, without resorting to any error mitigation techniques based on Richardson extrapolation. more...

In the 1980s CK Vul was identified with Nova Vul 1670, as a potential "old nova" that could be used to test the "hibernation" scenario in relation to classical and dwarf novae. Since that time a number of different classifications have been suggested and discarded, including a classical nova, a very-late thermal pulse, a sub-Chandrasekhar mass supernova, a diffusion-induced nova and the ingestion of planets by their host star. Most recently the object has been linked to the red novae, of which V838 Mon is the most prominent member. The behaviour of this class is most likely due to a merger in a binary star system. However the characteristics of the brightening events in the 1670s that led to it being noticed by 17th Century astronomers, and the subsequent evolution, are inconsistent with a merger of any main sequence or giant progenitors. We have reviewed the data to date, and combined that with ALMA Cycle 4 results mapping dusty cocoons, gaseous bubbles, a jet and a central disc-like structure, to propose a new cause of "Nova Vul 1670". We suggest that both the original observations nearly 350 years ago and the current configuration of the system are consistent with a merger between a white dwarf and a brown dwarf. The disruption of the brown dwarf caused the brightening events in 1670, 1671 and 1672. In the process the collision of brown dwarf material with the white dwarf surface caused nucleosynthesis leading to the peculiar isotopic ratios seen in the ejecta. The encounter disrupted the brown dwarf, which formed a disc that persists to this day. The disc rotation and white dwarf magnetic fields generated bipolar jet outflows, which in turn inflated the gaseous bubbles. Over the intervening centuries these have grown to be about 12 arcsec in extent, and are surrounded by dusty cocoons, in a structure reminiscent of asymmetric planetary nebulae and pre-PNe. Further work remains to be done on the gas dynamics, and proposed observations will attempt to measure the central object mass directly from a disc rotation curve. more...

Summary. Background: Most Ca2+ signaling research in platelets has relied solely on monitoring the cytosolic Ca2+ concentration ([Ca2+]cyt). Changes in [Ca2+]cyt constitute the net effect of Ca2+ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca2+ sequestration into the stores and Ca2+ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca2+ signaling difficult and subject to error. Objectives: To validate the use of the low-affinity Ca2+ indicator Fluo-5N to monitor the concentration of Ca2+ in the intracellular stores ([Ca2+]st) of human platelets as a first step in developing assays for a systems-level analysis of platelet Ca2+ signaling. Methods: Fluo-5N-loaded and Fura-2-loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca2+]cyt and [Ca2+]st. Results: Fluo-5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca2+ stores and to physiologic agonists. Ca2+ reuptake inhibitors and blockers of Ca2+ release channels had the expected effects on Fura-2 and Fluo-5N fluorescence. Agonist-evoked Ca2+ release was reversed by Ca2+ addition to the medium, and required intact Ca2+ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non-SERCA Ca2+ reuptake mechanism. Evidence for a role for Ca2+-induced Ca2+ release in agonist-evoked responses was obtained. Conclusions: Our data provide a validation of the use of Fluo-5N as a method for monitoring changes in [Ca2+]st in human platelets. more...

Summary. Background: Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca2+entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca2+entry in human platelets for Na+/Ca2+ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca2+entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca2+signaling in human platelets. Methods: Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca2+or Na+ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na+, significantly reduced PMA-evoked Ca2+entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P2X1 antagonists Ro-0437626 or MRS-2159, or desensitization of P2X1 receptors by prior treatment with α,β-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca2+entry. Ro-0437626 or chelation of extracellular Ca2+ slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P2X1 receptor activation but is accelerated by P2X1-mediated Ca2+entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA-evoked Ca2+entry results from an NCX3-dependent dense granule secretion and subsequent P2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway. more...

Summary. Background: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. Objectives: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. Methods: Platelets loaded with fura-2 or sodium-binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5-HT secretion was determined by fluorescence assay after conjugation with o-phthaldialdehyde. ATP secretion was determined using luciferin-luciferase. Results: TRPV1 was identified by Western blotting using a specific anti-hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca2+ influx and Ca2+ release from intracellular stores, responses that were blocked in a dose-dependent manner by the TRPV1 antagonists, 5′-Iodo-resiniferatoxin (5′-Iodo-RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na+]. Capsaicin-evoked Ca2+ release was abolished in the absence of extracellular Na+ or by the 5-HT2A receptor antagonist, ketanserin. Capsaicin evoked 5-HT release from platelets, a response abolished in the absence of extracellular Na+ or by 5′-Iodo-RTX. Thus capsaicin-evoked Ca2+ release appeared to be mediated by Na+-dependent 5-HT release. TRPV1-dependent 5-HT release also contributed to ADP- and thrombin-evoked Ca2+ entry and release. 5′-Iodo-RTX reduced ADP- and thrombin-evoked Ca2+ signals, effects not additive with those of ketanserin, and 5′-Iodo-RTX inhibited agonist-evoked 5-HT and ATP release. Conclusion: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis. more...

Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling. more...

Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling. more...

Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation. more...

Summary. Background: The role of the tyrosine kinase pp60src in PAR-1-dependent Ca2+ entry was investigated in human platelets. pp60src plays a role in thapsigargin (TG)-evoked store-operated Ca2+ entry (SOCE), which is thought to be a major component of thrombin-evoked Ca2+ entry. Methods: pp60src tyr416 phosphorylation was used to assess pp60src activation. Fura-2-loaded platelets were used to monitor intracellular Ca2+ concentration ([Ca2+]i). Results: Activation of PAR-1 with the specific agonist SFLLRN increased pp60src activation within 10 s. This required phospholipase C (PLC) activity, Ca2+ release and a rise in intracellular Ca2+. PP2, an inhibitor of Src-family tyrosine kinases, inhibited SFLLRN-evoked Ca2+ entry, but also inhibited Ca2+ release and the extrusion of Ca2+ by the plasma membrane Ca2+ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG- and SFLLRN-evoked pp60src activation. Although Go6976, an inhibitor of cPKCs, inhibited TG-evoked SOCE, it had little effect on SFLLRN- or thrombin-evoked Ca2+ entry. Conclusions: These data indicate that stimulation of PAR-1 leads to activation of pp60src in human platelets, through PLC and cPKC activation, Ca2+ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN-evoked Ca2+ entry, we suggest that pp60src is also not required. The apparent inhibition of SFLLRN-evoked Ca2+ entry by PP2 is likely to be secondary to reduced Ca2+ release. These data argue against a contribution of this SOCE pathway to PAR-1-dependent Ca2+ entry. more...

Platelet activation by potent, Ca(2+)-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from alphaIIbbeta3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo. more...

Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets. more...

Depletion of intracellular Ca2+ stores results in the activation of SMCE (store-mediated Ca2+ entry) in many cells. The mechanism of activation of SMCE is poorly understood. In human platelets, a secretion-like coupling model may be involved. This proposes that store depletion results in trafficking of portions of the endoplasmic reticulum to the plasma membrane, enabling coupling between proteins in the two membranes. In support of this, we have shown that, in human platelets, agonist-evoked Ca2+ store depletion results in de novo and reversible coupling of the InsP3RII [type II inositol (1,4,5)trisphosphate receptor] with the putative Ca2+ entry channel hTRPC1 [human canonical transient receptor potential 1 (protein); Rosado, Brownlow and Sage (2002) J. Biol. Chem. 277, 42157–42163]. A crucial test of the hypothesis that this coupling activates SMCE is that it should occur rapidly enough to account for agonist-evoked Ca2+ entry. In the present study, we have used quenched- and stopped-flow approaches to determine the latencies of thrombin-evoked coupling of InsP3RII with hTRPC1 and of thrombin-evoked bivalent cation entry using Mn2+ quenching of fura 2 fluorescence. Thrombin-evoked Mn2+ entry was detected with a latency of 0.81±0.07 s (S.E.M., n=7) or 1.36±0.09 s (S.E.M., n=7) at a concentration of 1.0 or 0.1 unit/ml respectively. Coupling between InsP3RII and hTRPC1, assessed at 100 ms intervals, was first detected with a latency of 0.9 or 1.4 s after stimulation with thrombin at a concentration of 1.0 or 0.1 unit/ml respectively. These results support the hypothesis that de novo coupling of InsP3RII with hTRPC1 could activate SMCE in human platelets. more...

Tumor necrosis factor-α (TNF-α) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-α on thapsigargin (TG)-induced store-mediated Ca2+entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-α slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-α significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-α abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-α was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-α inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-α. more...

Physical coupling between inositol 1,4,5-trisphosphate (IP3) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca2+ entry (SMCE). We have investigated the properties of the coupling between the type II IP3 receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP3 receptor function, abolished SMCE and coupling between the IP3 receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca2+ stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca2+ stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP3 receptors and hTrp1 in normal human cells. more...

1 In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. 2 Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. 3 Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. 4 Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. 5 Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. 6 These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores. more...

We have recently reported that store-mediated Ca2+ entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed ‘secretion-like coupling’. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca2+ entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca2+ entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca2+ entry. In addition, depletion of intracellular Ca2+ stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60src, a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca2+ entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca2+ entry in human platelets. more...

In the present study we have investigated the role of inositol 1,4, 5-trisphosphate (IP(3)), functional IP(3) receptors (IP(3)Rs) and the human homologue of the Drosophila transient receptor potential (Trp) channel, human Trp1 (hTrp1), in store-mediated Ca(2+) entry (SMCE) in human platelets. Inhibition of IP(3) recycling using Li(+), or the inhibition of IP(3)Rs using xestospongin C, both resulted in the inhibition of SMCE activation following Ca(2+) store depletion using thapsigargin. Co-immunoprecipitation experiments indicated that endogenously expressed hTrp1 couples with IP(3)R type II, but not types I or III, in platelets with depleted intracellular Ca(2+) stores, but not in control, undepleted cells. These results provide strong evidence for the activation of SMCE by conformational coupling involving de novo association between IP(3)Rs and a plasma membrane channel in normal human cells. more...

We have investigated the mechanism of Ca(2+) entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the presence, but not in the absence, of external Ca(2+), suggesting a relatively selective inhibition of Ca(2+) entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca(2+) entry evoked by the depletion of intracellular Ca(2+) stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca(2+) entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca(2+) entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca(2+) entry, indicating a cytoskeleton-independent component in the regulation of Ca(2+) entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca(2+) entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets. more...

The effects of osmotic stress on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of excised intact rat aorta were investigated using the indicator fura-2 and the patch clamp technique. Hyperosmotic stress evoked a reversible rise in endothelial [Ca2+]i in the presence but not absence of extracellular Ca2+, indicating that it evoked Ca2+ entry without release from intracellular stores. Hyposmotic stress was without significant effect. Cytochalasins B and D reduced the effect of hyperosmotic stress but not acetylcholine on endothelial [Ca2+]i. In endothelium isolated from underlying smooth muscle, hyperosmotic stress produced a rise in the [Ca2+]i and depolarisation of the endothelial membrane potential whereas hyposmotic stress was without effect. Mechanosensitive cationic channels recorded in cell-attached patches were activated by hyperosmotic solutions applied to the endothelium and inhibited by hyposmotic solutions. These data suggest that shrinkage of endothelial cells evokes an increase in [Ca2+]i by opening a pathway for Ca2+ entry from the extracellular space. The mechanosensitive ionic channels which we have previously described may be responsible for this response. more...