Ca2(+)-driven [3H]arachidonate release in electropermeabilized human platelets shows an absolute requirement for MgATP2-.
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ABSTRACT:
SUBMITTER: Patel S
PROVIDER: S-EPMC1149799 | biostudies-other | 1991 Feb
REPOSITORIES: biostudies-other
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Project description:The effect of extracellular Na+ removal and replacement with other cations on receptor-mediated arachidonate release in platelets was studied to investigate the role of Na+/H+ exchange in this process. Replacement with choline+, K+, N-methylglucamine+ (which abolished the thrombin-induced pHi rise) or Li+ (which allowed a normal thrombin-induced pHi rise) significantly decreased arachidonate release in response to all concentrations (threshold to supra-maximal) of thrombin and collagen. This inhibition was not reversed by NH4Cl (10 mM) addition, which raised the pHi in the absence of Na+, but, on the contrary, NH4Cl addition further decreased the extent of thrombin- and collagen-induced arachidonate release, as well as decreasing 'weak'-agonist (ADP, adrenaline)-induced release and granule secretion in platelet-rich plasma. No detectable pHi rises were seen with collagen (1-20 micrograms/ml) and ADP (10 microM) in bis-(carboxyethyl)carboxyfluorescein-loaded platelets. Inhibition of thrombin-induced pHi rises was seen with 0.5-5 microM-5-NN-ethylisopropylamiloride (EIPA), but at these concentrations EIPA had little effect on thrombin-induced arachidonate release. At higher concentrations such as those used in previous studies (20-50 microM), EIPA inhibited aggregation/release induced by collagen and ADP in Na+ buffer as well as in choline+ buffer (where there was no detectable exchanger activity), suggesting that these concentrations of EIPA exert 'non-specific' effects at the membrane level. The results suggest that (i) Na+/H+ exchange and pHi elevations are not only necessary, but are probably inhibitory, to receptor-mediated arachidonate release in platelets, (ii) inhibition of receptor-mediated release in the absence of Na+ is most likely due to the absent Na+ ion itself, and (iii) caution should be exercised in the use of compounds such as EIPA, which, apart from inhibiting the Na+/H+ exchanger, have other undesirable and misleading effects in platelets.
| S-EPMC1136625 | biostudies-other
Project description:Venous thromboembolic (VTE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils; however, the molecular mechanisms of neutrophil death in VTE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil-platelet aggregate formation induced by tumor necrosis factor-? but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space.
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Project description:Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.
| S-EPMC1137430 | biostudies-literature
Project description:The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.
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Project description:The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.
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Project description:Platelet stimulation by thrombin or Ca2+ ionophore induces mobilization of arachidonate from lipid stores. We have previously shown that, in [14C]arachidonic acid-prelabelled resting platelets, [14C]arachidonate was transferred from diacyl-sn-glycerophosphocholine to ethanolamine and choline-containing ether phospholipids. This transfer reached an equilibrium after 5 h incubation [Colard, Breton & Bereziat (1984a) Biochem. J. 222, 657-662]. [14C]Arachidonate-prelabelled platelets having reached this transfer equilibrium were used to study the mobilization of arachidonate in etheracyl and diacyl phospholipids. Upon thrombin stimulation, arachidonate decreased in diacyl-sn-glycero-3-phosphoinositol, in alkylacyl- and diacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl- and diacyl-sn-glycero-3-phosphoethanolamine. Upon challenge with Ca2+ ionophore A23187, arachidonate decreased in diacyl-sn-glycero-3-phosphoethanolamine, in diacyl- and alkylacyl-sn-glycero-3-phosphocholine and increased in alkenylacyl-sn-glycero-3-phosphoethanolamine. We also compared arachidonate mobilization in platelets stimulated immediately after [14C]arachidonic acid chase with platelets stimulated after 5 h reincubation. We observed that the arachidonate newly incorporated into diacyl-sn-glycero-3-phosphocholine and triacylglycerols was rapidly released upon stimulation. This suggests the presence in these two lipids of a rapidly-turning-over arachidonate pool.
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Project description:Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group.
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Project description:How morphogen gradients are shaped is a major question in developmental biology, but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to Hh N-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand.
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Project description:In cardiac mitochondria, matrix free Ca(2+) ([Ca(2+)]m) is primarily regulated by Ca(2+) uptake and release via the Ca(2+) uniporter (CU) and Na(+)/Ca(2+) exchanger (NCE) as well as by Ca(2+) buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca(2+) buffering affects these dynamics under various Ca(2+) uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca(2+) buffering on the uptake and release of Ca(2+), we monitored Ca(2+) dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca(2+)] ([Ca(2+)]e) and [Ca(2+)]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca(2+)]e and [Ca(2+)]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca(2+)-induced changes in mitochondrial bioenergetics. Our [Ca(2+)]e and [Ca(2+)]m measurements demonstrate that Ca(2+) uptake and release do not show reciprocal Ca(2+) dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca(2+) buffering system in the matrix compartment. The Na(+)- induced Ca(2+) release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca(2+) uptake in the presence of large amounts of CaCl2, but not by Na(+)- induced Ca(2+) release.
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