FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012347765> ?p ?o ?g. }

• W2012347765 endingPage "29235" @default.
• W2012347765 startingPage "29231" @default.
• W2012347765 abstract "A major pathway for stimulated Ca2+ entry in non-excitable cells is activated following depletion of intracellular Ca2+ stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca2+ stores has been proposed as the most likely mechanism to activate this store-mediated Ca2+ entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca2+ pools, which are differentially modulated by the actin cytoskeleton. Ca2+ entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca2+ entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca2+ entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca2+ entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM. A major pathway for stimulated Ca2+ entry in non-excitable cells is activated following depletion of intracellular Ca2+ stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca2+ stores has been proposed as the most likely mechanism to activate this store-mediated Ca2+ entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca2+ pools, which are differentially modulated by the actin cytoskeleton. Ca2+ entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca2+ entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca2+ entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca2+ entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM. SMCE 1The abbreviations used are: SMCE, store-mediated calcium entry; [Ca2+]i, intracellular free calcium concentration; TG, thapsigargin; Iono, ionomycin; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; IP3, inositol 1,4,5-trisphosphate; PM, plasma membrane; CytD, cytochalasin D; Lat A, latrunculin A; FTA, farnesylthioacetic acid; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; JP, jasplakinolide; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase.1The abbreviations used are: SMCE, store-mediated calcium entry; [Ca2+]i, intracellular free calcium concentration; TG, thapsigargin; Iono, ionomycin; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; IP3, inositol 1,4,5-trisphosphate; PM, plasma membrane; CytD, cytochalasin D; Lat A, latrunculin A; FTA, farnesylthioacetic acid; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; JP, jasplakinolide; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase. is triggered by depletion of the intracellular Ca2+ stores (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2086) Google Scholar), although the mechanism underlying this process is not fully understood. Several hypotheses have been proposed to account for the communication between the intracellular Ca2+ stores and the PM, which can be grouped into two main categories: the conformational or secretion-like coupling hypotheses, which propose physical coupling between elements in the Ca2+ stores and the PM, and diffusible messenger hypotheses, which propose the release of a small molecule from the Ca2+ stores that opens, directly or indirectly, Ca2+ channels in the PM (2Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1285) Google Scholar).Recently, the secretion-like coupling model has received support from studies showing that activation of SMCE shares properties with the activation of secretion (3Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) In several non-excitable cells, including platelets, actin filament reorganization plays a key role in the activation of SMCE, perhaps by facilitating translocation of Ca2+ stores to the PM to enable coupling (3Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 5Mehta D. Ahmmed G.U. Paria B.C. Holinstat M. Voyno-Yasenetskaya T. Tiruppathi C. Minshall R.D. Malik A.B. J. Biol. Chem. 2003; 278: 33492-33500Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Redondo P.C. Lajas A.I. Salido G.M. Gonzalez A. Rosado J.A. Pariente J.A. Biochem. J. 2003; 370: 255-263Crossref PubMed Scopus (55) Google Scholar, 7Rosado J.A. Rosenzweig I. Harding S. Sage S.O. Am. J. Physiol. 2001; 280: C1636-C1644Crossref Google Scholar). In human platelets, where we have demonstrated a secretion-like coupling mechanism, Ca2+ entry is proposed to be based on reversible trafficking of portions of the Ca2+ stores toward the PM to facilitate de novo coupling between the type II inositol 1,4,5-trisphosphate (IP3) receptor in the store membrane and naturally expressed human canonical transient receptor potential 1 (hTRPC1) in the PM (8Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, 9Rosado J.A. Sage S.O. Biochem. J. 2001; 356: 191-198Crossref PubMed Scopus (106) Google Scholar, 10Rosado J.A. Redondo P.C. Salido G.M. Gomez-Arteta E. Sage S.O. Pariente J.A. J. Biol. Chem. 2004; 279: 1665-1675Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In this process the actin cytoskeleton acts as a negative modulator of the interaction between the Ca2+ store and PM but is also required for activation of SMCE, since cytoskeletal disruption impairs Ca2+ entry (4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).Platelets express at least two isoforms of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), with molecular masses of 100 and 97 kDa (11Papp B. Enyedi A. Kovacs T. Sarkadi B. Wuytack F. Thastrup O. Gardos G. Bredoux R. Levy-Toledano S. Enouf J. J. Biol. Chem. 1991; 266: 14593-14596Abstract Full Text PDF PubMed Google Scholar). The 100-kDa isoform, which is inhibited by low concentrations of TG, has been identified as SERCA 2b. The 97-kDa isoform is much less sensitive to TG (11Papp B. Enyedi A. Kovacs T. Sarkadi B. Wuytack F. Thastrup O. Gardos G. Bredoux R. Levy-Toledano S. Enouf J. J. Biol. Chem. 1991; 266: 14593-14596Abstract Full Text PDF PubMed Google Scholar) and, unlike the 100-kDa isoform, is inhibited by 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (12Papp B. Enyedi A. Paszty K. Kovacs T. Sarkadi B. Gardos G. Magnier C. Wuytack F. Enouf J. Biochem. J. 1992; 288: 297-302Crossref PubMed Scopus (89) Google Scholar). This has been identified as SERCA 3 (13Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Bobe R. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar). Pharmacological studies suggest that these two SERCA isoforms are distributed separately in discrete subpopulations of the IP3-sensitive Ca2+ stores (14Cavallini L. Coassin M. Alexandre A. Biochem. J. 1995; 310: 449-452Crossref PubMed Scopus (78) Google Scholar). TBHQ or high concentrations of TG release Ca2+ from one pool (TBHQ-sensitive store with low affinity for TG), while low (about 10 nm) concentrations of TG release Ca2+ from a different compartment (TBHQ-insensitive store with high affinity for TG) (14Cavallini L. Coassin M. Alexandre A. Biochem. J. 1995; 310: 449-452Crossref PubMed Scopus (78) Google Scholar). Immunolocalization studies indicate that the SERCAs 2b and 3 have different distributions in platelets (15Kovacs T. Berger G. Corvazier E. Paszty K. Brown A. Bobe R. Papp B. Wuytack F. Cramer E.M. Enouf J. Br. J. Haematol. 1997; 97: 192-203Crossref PubMed Scopus (35) Google Scholar), although these have not been clearly resolved.Here we show that SMCE in platelets is the result of the combined effects of depletion of two different Ca2+ stores and that these two pathways are differentially modulated by the actin cytoskeleton.EXPERIMENTAL PROCEDURESMaterials—fura-2 acetoxymethyl ester and jasplakinolide were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin, and bovine serum albumin were from Sigma (Madrid, Spain). Ionomycin (Iono), cytochalasin D (CytD), and latrunculin A (Lat A) were from Calbiochem (Nottingham, UK). Farnesylthioacetic acid (FTA), acetylgeranylgeranyl cysteine (AGGC), and TBHQ were from Alexis (Nottingham, UK). All other reagents were of analytical grade.Platelet Preparation—fura-2-loaded platelets were prepared as described previously (4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Briefly, blood was obtained from healthy drug-free volunteers and mixed with ⅙ volume of acid/citrate dextrose anti-coagulant containing (in mm): 85 sodium citrate, 78 citric acid, and 111 d-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 × g, and aspirin (100 μm) and apyrase (40 μg/ml) were added. Platelet-rich plasma was incubated at 37 °C with 2 μm fura-2 acetoxymethyl ester for 45 min. Cells were then collected by centrifugation at 350 × g for 20 min and resuspended in Hepes-buffered saline, pH 7.45, containing (in mm): 145 NaCl, 10 Hepes, 10 d-glucose, 5 KCl, 1 MgSO4 and supplemented with 0.1% bovine serum albumin and 40 μg/ml apyrase.Measurement of Intracellular Free Calcium Concentration ([Ca2+]i)— Fluorescence was recorded from 2-ml aliquots of magnetically stirred platelet suspension (2 × 108 cells/ml) at 37 °C using a Cary Eclipse Spectrophotometer (Varian Ltd., Madrid, Spain) or a Cairn Research Spectrofluorimeter (Cairn Research Ltd., Faversham, UK) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca2+]i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. (16Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar).Ca2+ entry was estimated using the integral of the rise in [Ca2+]i for 2.5 min after addition of CaCl2 (4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Ca2+ release was estimated using the integral of the rise in [Ca2+]i for 9 min, for TBHQ, or 2.5 min TG or TG + Iono, after Ca2+ or agonist addition. Both Ca2+ entry and release are expressed as nm, as described previously (17Heemskerk J.W. Feijge M.A. Henneman L. Rosing J. Hemker H.C. Eur. J. Biochem. 1997; 249: 547-555Crossref PubMed Scopus (86) Google Scholar, 18Rosado J.A. Sage S.O. Biochem. J. 2000; 346: 183-192Crossref Google Scholar). When platelets were preincubated with inhibitors, Ca2+ entry was corrected by subtraction of the [Ca2+]i elevation due to leakage of the indicator.To investigate SMCE induced by depletion of TBHQ-sensitive and -insensitive stores individually we followed the procedure depicted in Fig. 2. This protocol consists of depleting the TBHQ-sensitive store to investigate the subsequently induced Ca2+ entry, followed by depletion of the TBHQ-insensitive store using TG + Iono (Fig. 2, B and E) to monitor Ca2+ influx, in this case due to depletion of both stores. Complete depletion of the TBHQ-insensitive store by TG alone required a long exposure to this agent, which might lead to deterioration of the cells; therefore we used TG (100 nm) combined with a low concentration of Iono (20 nm) to induce rapid store depletion. Ca2+ entry activated by the TBHQ-insensitive pool was estimated by subtraction of the increase in [Ca2+] after the addition of Ca2+i to the medium in platelets not stimulated with TG + Iono (Fig. 2, A and D).RESULTSWe have previously shown that treatment of platelets with TG in a Ca2+-free medium results in a sustained increase in [Ca2+]i due to Ca2+ release from intracellular stores. Subsequent addition of Ca2+ to the extracellular medium induces a prolonged elevation in [Ca2+]i indicative of SMCE (4Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 7Rosado J.A. Rosenzweig I. Harding S. Sage S.O. Am. J. Physiol. 2001; 280: C1636-C1644Crossref Google Scholar, 8Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar). SMCE induced by a short (3 min) treatment with TG was significantly reduced by treatment for 40 min with the actin filament disrupter CytD (10 μm), which abolishes actin polymerization in platelets (18Rosado J.A. Sage S.O. Biochem. J. 2000; 346: 183-192Crossref Google Scholar).As shown in Fig. 1A, SMCE induced by longer exposures to TG (200 nm) is less sensitive to the disassembly of the actin cytoskeleton, and this modification even increases SMCE activated by a 30-min treatment with TG. We also compared the effect of CytD on SMCE induced by treatment with TG (100 nm) alone or together with a low concentration of Iono (20 nm) to accelerate depletion of the Ca2+ stores. Treatment of platelets with CytD did not significantly modify Ca2+ release induced by TG alone or in combination with Iono, suggesting that Ca2+ accumulation is not affected by disruption of the actin cytoskeleton (Fig. 1B; n = 6). In CytD-treated platelets TG-induced SMCE was significantly reduced compared with controls (Fig. 1C; p < 0.05; n = 6), whereas SMCE induced by TG + Iono was increased by treatment with CytD (Fig. 1C; p < 0.05; n = 6).Fig. 1The effect of cytochalasin D on SMCE depends on the extent of store depletion. A, fura-2-loaded human platelets were preincubated for 40 min at 37 °C with 10 μm CytD or the vehicle (Me2SO) and then treated in a Ca2+-free medium (200 μm EGTA was added) for various periods of time with TG (200 nm) followed by addition of CaCl2 (final concentration: 300 μm) to initiate Ca2+ entry. Values indicate the percentage of Ca2+ entry under the different experimental conditions relative to their control and are expressed as average ± S.E. from eight to eleven separate experiments. B and C, human platelets were preincubated for 40 min at 37 °C with 10 μm CytD or the vehicle (Me2SO) and then treated in a Ca2+-free medium (200 μm EGTA was added) with TG (100 nm) or TG (100 nm) + Iono (20 nm) followed by addition of CaCl2 (final concentration: 300 μm) 3 min later to initiate Ca2+ entry. Values indicate the percentage of Ca2+ release (B) and entry (C) under the different experimental conditions relative to their control and are expressed as average ± S.E. from six separate experiments. *, p < 0.05 compared with control.View Large Image Figure ViewerDownload (PPT)There are at least two possible explanations for these observations. Either different Ca2+ entry pathways, which are differentially modulated by the actin cytoskeleton, are activated according to the extent of Ca2+ store depletion, or depletion of different intracellular Ca2+ stores gives rise to the activation of two Ca2+ entry pathways. In the latter case, a short treatment with TG would be expected to affect mainly the Ca2+ stores with high TG affinity, while a prolonged exposure to TG might deplete Ca2+ pools with both high and low affinity for TG.To further investigate this phenomenon we investigated the effects of sequential application of two different SERCA inhibitors, TBHQ and TG. Treatment of platelets with TBHQ induced a concentration-dependent Ca2+ release from a TBHQ-sensitive component of the intracellular Ca2+ stores, reaching a maximal effect at concentrations of 20 μm (data not shown). Ca2+ entry was also maximal after treatment with 20 μm TBHQ (not shown); therefore we used 20 μm TBHQ throughout this study.Treatment of platelets in a Ca2+-free medium (300 μm EGTA was added) with TBHQ (20 μm; Fig. 2) induced a sustained increase in [Ca2+]i; subsequent addition of CaCl2 (500 μm) 9 min later induced a larger, prolonged increase in [Ca2+]i indicative of Ca2+ entry (Fig. 2). The extracellular Ca2+ was chelated 3 min later by addition of EGTA (1 mm), and cells were then treated with TG (100 nm) + Iono (20 nm; Fig. 2, B and E) for a further 5 min or with the vehicle (Me2SO; Fig. 2, A and D). Treatment with TG + Iono induced a transient increase in [Ca2+]i due to Ca2+ release from a TBHQ-insensitive component of the Ca2+ stores. The amount of Ca2+ released by TG + Iono was 5-fold greater than that released by TBHQ, suggesting that the TBHQ-releasable component of the pool is smaller in size than the TBHQ-insensitive component. The subsequent addition of CaCl2 (1.3 mm) resulted in a rise in [Ca2+]i 6-fold higher than that observed when the TBHQ-sensitive component of the stores alone were emptied (Fig. 2, B and E; p < 0.05; n = 6–11). This difference cannot be attributed to a different extracellular Ca2+ concentration ([Ca2+]o), since a similar [Ca2+]o was present at the time when SMCE was initiated after depletion of one or both stores, as estimated using an established EGTA/Ca2+ buffer equation (19Tiffert T. Lew V. J. Physiol. 1997; 500: 139-154Crossref PubMed Scopus (36) Google Scholar). This is supported by the similar Ca2+ entries observed on each [Ca2+]o elevation in Fig. 2, A and D, in the absence of TG + Iono.Treatment of human platelets with CytD (10 μm for 40 min) or Lat A (3 μm for 1 h), two unrelated actin polymerization inhibitors, increased TBHQ-induced SMCE by 331 ± 66% and 411 ± 30%, respectively (Fig. 2, A–F; p < 0.05; n = 6). In contrast, SMCE induced following the combined actions of TBHQ, TG, and Iono was reduced (Fig. 2, B, C, E, and F). SMCE induced by the TBHQ-insensitive component of the stores alone (see “Experimental Procedures”) was reduced by 40 ± 7 and 70 ± 2% in CytD- and Lat A-treated cells, respectively.Statistical Analysis—Analysis of statistical significance was performed using Student's t test. p < 0.05 was considered to be significant for a difference.These results suggest against differential depletion of a single intracellular Ca2+ store being responsible for the activation of two pathways for SMCE, which are regulated differently by the actin cytoskeleton. SMCE activated following a small degree of Ca2+ store depletion by treatment with TBHQ was potentiated by disruption of the actin cytoskeleton (Fig. 2, A–F), whereas cytoskeletal disruption inhibited SMCE activated following brief treatments with TG (Fig. 1, A and C); potentiation was only observed after more extensive store depletion due to longer exposures to TG (Fig. 1A) or the combined actions of Iono and TG (Fig. 1C). Rather, these results support the hypothesis that two mechanisms for SMCE that are differently regulated by the actin cytoskeleton are activated following depletion of two discrete Ca2+ stores, one with a low sensitivity to TG but sensitive to TBHQ and another that is insensitive to TBHQ but highly sensitive to TG. Thus these two Ca2+ entry pathways appear to be activated by the depletion of the two intracellular Ca2+ stores identified by Cavallini et al. (14Cavallini L. Coassin M. Alexandre A. Biochem. J. 1995; 310: 449-452Crossref PubMed Scopus (78) Google Scholar).We further explored this issue by testing the involvement of the Ras superfamily of proteins in the two pathways for SMCE. Ras family proteins are required for the activation of SMCE in platelets (18Rosado J.A. Sage S.O. Biochem. J. 2000; 346: 183-192Crossref Google Scholar) and other cells (20Bird G.S. Putney J.W. J. Biol. Chem. 1993; 268: 21486-21488Abstract Full Text PDF PubMed Google Scholar). In platelets this mechanism involves the reorganization of the actin cytoskeleton. To investigate the involvement of Ras family proteins we used a combination of FTA and AGGC, inhibitors of methylation, and thus activation of prenylated and geranylgeranylated Ras family proteins (18Rosado J.A. Sage S.O. Biochem. J. 2000; 346: 183-192Crossref Google Scholar, 21Xu Y. Gilbert B.A. Rando R.R. Chen L. Tashjian Jr., A.H. Mol. Pharmacol. 1996; 50: 1495-1501PubMed Google Scholar). As observed previously with the actin cytoskeleton disrupters, treatment of platelets for 30 min with 40 μm FTA combined with 30 μm AGGC increased TBHQ-induced SMCE by 350 ± 72% (Fig. 3; p < 0.05; n = 8). In contrast, total SMCE induced by depletion of both stores was clearly reduced. SMCE induced by the TBHQ-insensitive store alone was reduced by 52 ± 6% (Fig. 3). As expected, since Ras family proteins participate in the maintenance and the dynamics of the cytoskeletal structure, the effect of these proteins on both Ca2+ entry models is similar to that observed following disruption of the actin cytoskeleton.Fig. 3Role of Ras family proteins in Ca2+ entry induced by depletion of TBHQ-sensitive and -insensitive stores. fura-2-loaded human platelets were preincubated for 30 min at 37 °C with 40 μm FTA plus 30 μm AGGC or the vehicle (Me2SO) as control (bold traces) and then treated in a Ca2+-free medium (300 μm EGTA was added) for 9 min with TBHQ (20 μm) followed by addition of CaCl2 (500 μm) to initiate Ca2+ entry. Extracellular Ca2+ was chelated again by addition of EGTA (final concentration: 1 mm), and then platelets were stimulated in the absence (A) or presence of TG (100 nm) plus Iono (20 nm; B) followed by the addition of CaCl2 (1.3 mm) to allow Ca2+ entry. C, histograms indicate the percentage of Ca2+ entry under the different experimental conditions relative to their control (vehicle was added). Ca2+ entry was determined as described under “Experimental Procedures,” and values are expressed as average ± S.E. from eight independent experiments. *, p < 0.05 compared with control.View Large Image Figure ViewerDownload (PPT)In the “secretion-like coupling” hypothesis, the cortical actin network acts as a clamp that blocks the interaction between the Ca2+ stores and PM; therefore disorganization of the actin cytoskeleton would be expected to facilitate Ca2+ entry. Consistent with this, we have observed an increase in the amount of Ca2+ entry when this event was initiated by depletion of the TBHQ-sensitive stores. In contrast, when the TBHQ-insensitive pool was emptied, actin-depolymerizing agents reduced Ca2+ entry, suggesting that the actin cytoskeleton also plays a positive role in the activation of this mechanism.To investigate whether the membrane-associated cytoskeleton acts as a physical barrier that prevents Ca2+ entry, we used jasplakinolide (JP), a cell-permeant peptide that induces polymerization and redistribution of actin filaments into a thick cortical layer subjacent to the PM (3Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 22Rosado J.A. Sage S.O. J. Physiol. (Lond.). 2000; 526: 221-229Crossref Scopus (136) Google Scholar). Interestingly, treatment of platelets for 30 min with 10 μm JP significantly reduced SMCE induced by depletion of TBHQ-sensitive and -insensitive stores by 50% (Fig. 4; p < 0.05; n = 5). These observations suggest that the cortical actin cytoskeleton exerts a negative regulatory role on both SMCE pathways, perhaps preventing the coupling between IP3 receptors in the Ca2+ stores and TRP channels in the PM as previously reported (8Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, 9Rosado J.A. Sage S.O. Biochem. J. 2001; 356: 191-198Crossref PubMed Scopus (106) Google Scholar).Fig. 4Effect of jasplakinolide on Ca2+ entry induced by depletion of TBHQ-sensitive and -insensitive stores. fura-2-loaded human platelets were preincubated for 30 min at 37 °C with 10 μm JP or the vehicle (Me2SO) as control (bold traces) and then treated in a Ca2+-free medium (300 μm EGTA was added) for 9 min with TBHQ (20 μm) followed by addition of CaCl2 (500 μm) to initiate Ca2+ entry. Extracellular Ca2+ was chelated again by addition of EGTA (final concentration: 1 mm), and then platelets were stimulated in the absence (A) or presence of TG (100 nm) plus Iono (20 nm; B) followed by the addition of CaCl2 (1.3 mm) to allow Ca2+ entry. C, histograms indicate the percentage of Ca2+ entry under the different experimental conditions relative to their control (vehicle was added). Ca2+ entry was determined as described under “Experimental Procedures,” and values are expressed as average ± S.E. from five independent experiments. *, p < 0.05 compared with control.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe existence of two Ca2+ stores and their differentially regulated Ca2+ entry pathways in human platelets requires a reinterpretation of the previously described model for SMCE in these cells. We believe that the secretion-like coupling model explains Ca2+ entry induced by the TBHQ-insensitive store, since it shows high affinity for TG and must be the store affected by short treatments with TG. According to this model, and consistent with the results presented here, the actin cytoskeleton plays a dual role in the activation of SMCE by this pathway: a positive role as a support for the transport of portions of the Ca2+ store to the proximity of the PM to allow coupling to occur, which is impaired by cytoskeletal disrupters, and a negative effect provided by the membrane-associated cytoskeleton to prevent constitutive activation of Ca2+ entry (Fig. 5). In addition, we have described for the first time a new SMCE pathway controlled by a TBHQ-sensitive pool with low affinity for TG where the membrane cytoskeleton acts only as a physical actin barrier to prevent coupling between the Ca2+ stores and PM under resting conditions (Fig. 5). The effect observed with JP suggests that for both SMCE pathways, the cortical actin cytoskeleton must be reorganized when the stores are depleted to facilitate the approach of the Ca2+ stores to the PM. The distinct regulation of both mechanisms might be explained by the stores having different locations. The TBHQ-sensitive store might be close enough to the PM for disruption of the membrane cytoskeleton alone to allow coupling. Coupling must only occur after store depletion however, since disruption of the actin cytoskeleton alone using CytD does not induce this event (9Rosado J.A. Sage S.O. Biochem. J. 2001; 356: 191-198Crossref PubMed Scopus (106) Google Scholar).Fig. 5Two pathways for SMCE in human platelets. We propose the existence of two mechanisms for SMCE induced by depletion of TBHQ-sensitive and -insensitive stores in platelets as differentiated by the role of the actin cytoskeleton. For Ca2+ entry induced by depletion of TBHQ-sensitive stores, which might be in close apposition with the plasma membrane, the actin filament network acts just as a barrier preventing constitutive coupling between Ca2+ channels in the plasma membrane and IP3 receptors in the stores and therefore Ca2+ influx. On the other hand, the actin cytoskeleton plays a dual role in Ca2+ entry induced by depletion of the TBHQ-insensitive pool, acting both as a barrier against constitutive coupling and a support for vesicle trafficking toward the plasma membrane.View Large Image Figure ViewerDownload (PPT)This new two-store model for SMCE explains the different effects of CytD on platelets treated for different periods of time with TG or with TG ± Iono. After short treatments with TG alone, SMCE is activated by the TBHQ-insensitive store and therefore is reduced by CytD. When Iono was added together with TG, or after long exposures to TG, both stores were emptied and a combination of effects was observed, and the CytD-induced inhibition of SMCE initiated by the TBHQ-insensitive store is compensated for by the enhanced SMCE activated by the TBHQ-sensitive pool. SMCE 1The abbreviations used are: SMCE, store-mediated calcium entry; [Ca2+]i, intracellular free calcium concentration; TG, thapsigargin; Iono, ionomycin; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; IP3, inositol 1,4,5-trisphosphate; PM, plasma membrane; CytD, cytochalasin D; Lat A, latrunculin A; FTA, farnesylthioacetic acid; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; JP, jasplakinolide; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase.1The abbreviations used are: SMCE, store-mediated calcium entry; [Ca2+]i, intracellular free calcium concentration; TG, thapsigargin; Iono, ionomycin; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; IP3, inositol 1,4,5-trisphosphate; PM, plasma membrane; CytD, cytochalasin D; Lat A, latrunculin A; FTA, farnesylthioacetic acid; AGGC, N-acetyl-S-geranylgeranyl-l-cysteine; JP, jasplakinolide; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase. is triggered by depletion of the intracellular Ca2+ stores (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2086) Google Scholar), although the mechanism underlying this process is not fully understood. Several hypotheses have been proposed to account fo" @default.
• W2012347765 created "2016-06-24" @default.
• W2012347765 creator A5002282410 @default.
• W2012347765 creator A5005766947 @default.
• W2012347765 creator A5030715778 @default.
• W2012347765 creator A5036708850 @default.
• W2012347765 creator A5046366404 @default.
• W2012347765 creator A5051674131 @default.
• W2012347765 creator A5075722475 @default.
• W2012347765 creator A5080498077 @default.
• W2012347765 date "2004-07-01" @default.
• W2012347765 modified "2023-10-18" @default.
• W2012347765 title "Two Pathways for Store-mediated Calcium Entry Differentially Dependent on the Actin Cytoskeleton in Human Platelets" @default.
• W2012347765 cites W1545068312 @default.
• W2012347765 cites W1586649084 @default.
• W2012347765 cites W1613210651 @default.
• W2012347765 cites W1898648621 @default.
• W2012347765 cites W1966050211 @default.
• W2012347765 cites W1977613097 @default.
• W2012347765 cites W1995515664 @default.
• W2012347765 cites W2006239419 @default.
• W2012347765 cites W2027094257 @default.
• W2012347765 cites W2041690080 @default.
• W2012347765 cites W2049647579 @default.
• W2012347765 cites W2054675070 @default.
• W2012347765 cites W2070832223 @default.
• W2012347765 cites W2082127635 @default.
• W2012347765 cites W2082366270 @default.
• W2012347765 cites W2141260882 @default.
• W2012347765 cites W2152078010 @default.
• W2012347765 cites W2206236475 @default.
• W2012347765 cites W2272261055 @default.
• W2012347765 cites W2412223496 @default.
• W2012347765 cites W4249375250 @default.
• W2012347765 doi "https://doi.org/10.1074/jbc.m403509200" @default.
• W2012347765 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15136566" @default.
• W2012347765 hasPublicationYear "2004" @default.
• W2012347765 type Work @default.
• W2012347765 sameAs 2012347765 @default.
• W2012347765 citedByCount "88" @default.
• W2012347765 countsByYear W20123477652012 @default.
• W2012347765 countsByYear W20123477652013 @default.
• W2012347765 countsByYear W20123477652014 @default.
• W2012347765 countsByYear W20123477652015 @default.
• W2012347765 countsByYear W20123477652016 @default.
• W2012347765 countsByYear W20123477652017 @default.
• W2012347765 countsByYear W20123477652018 @default.
• W2012347765 countsByYear W20123477652019 @default.
• W2012347765 countsByYear W20123477652020 @default.
• W2012347765 crossrefType "journal-article" @default.
• W2012347765 hasAuthorship W2012347765A5002282410 @default.
• W2012347765 hasAuthorship W2012347765A5005766947 @default.
• W2012347765 hasAuthorship W2012347765A5030715778 @default.
• W2012347765 hasAuthorship W2012347765A5036708850 @default.
• W2012347765 hasAuthorship W2012347765A5046366404 @default.
• W2012347765 hasAuthorship W2012347765A5051674131 @default.
• W2012347765 hasAuthorship W2012347765A5075722475 @default.
• W2012347765 hasAuthorship W2012347765A5080498077 @default.
• W2012347765 hasBestOaLocation W20123477651 @default.
• W2012347765 hasConcept C125705527 @default.
• W2012347765 hasConcept C142669718 @default.
• W2012347765 hasConcept C1491633281 @default.
• W2012347765 hasConcept C178790620 @default.
• W2012347765 hasConcept C185592680 @default.
• W2012347765 hasConcept C203014093 @default.
• W2012347765 hasConcept C2993400109 @default.
• W2012347765 hasConcept C519063684 @default.
• W2012347765 hasConcept C55493867 @default.
• W2012347765 hasConcept C86803240 @default.
• W2012347765 hasConcept C89560881 @default.
• W2012347765 hasConcept C95444343 @default.
• W2012347765 hasConceptScore W2012347765C125705527 @default.
• W2012347765 hasConceptScore W2012347765C142669718 @default.
• W2012347765 hasConceptScore W2012347765C1491633281 @default.
• W2012347765 hasConceptScore W2012347765C178790620 @default.
• W2012347765 hasConceptScore W2012347765C185592680 @default.
• W2012347765 hasConceptScore W2012347765C203014093 @default.
• W2012347765 hasConceptScore W2012347765C2993400109 @default.
• W2012347765 hasConceptScore W2012347765C519063684 @default.
• W2012347765 hasConceptScore W2012347765C55493867 @default.
• W2012347765 hasConceptScore W2012347765C86803240 @default.
• W2012347765 hasConceptScore W2012347765C89560881 @default.
• W2012347765 hasConceptScore W2012347765C95444343 @default.
• W2012347765 hasIssue "28" @default.
• W2012347765 hasLocation W20123477651 @default.
• W2012347765 hasOpenAccess W2012347765 @default.
• W2012347765 hasPrimaryLocation W20123477651 @default.
• W2012347765 hasRelatedWork W1908941310 @default.
• W2012347765 hasRelatedWork W197432475 @default.
• W2012347765 hasRelatedWork W1989836623 @default.
• W2012347765 hasRelatedWork W1998274488 @default.
• W2012347765 hasRelatedWork W2056665186 @default.
• W2012347765 hasRelatedWork W2099875681 @default.
• W2012347765 hasRelatedWork W2594707584 @default.
• W2012347765 hasRelatedWork W2904912999 @default.
• W2012347765 hasRelatedWork W4303449254 @default.
• W2012347765 hasRelatedWork W4311957677 @default.
• W2012347765 hasVolume "279" @default.

• next