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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
B, and Activator Protein 1 in T Lymphocytes
Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, Spain (C.N., R.S., F.J.C., E.M.); Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale, Novara, Italy (F.P., G.A.); Vivacell Biotechnology GmbH, Denzlingen, Germany (B.L.F.); and Department of Organic Chemistry, Lund University, Lund, Sweden (O.S.)
Received May 20, 2006; accepted July 12, 2006.
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B
degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-B inhibitory protein and to activate the c-Jun NH2-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor- gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.
). TG causes a net transfer of Ca2+ from the endoplasmic reticulum (ER) to cytosol, elevating [Ca2+]i (Thastrup et al., 1990; Rooney and Meldolesi, 1996) and inducing apoptosis via ER stress. We have recently shown that, apart from TG, T. garganica also contains compounds capable of mobilizing Ca2+ from the ER. These were identified in a series of unique C19 dilactones named basiliolides (Appendino et al., 2005).
In biological systems, calcium ions (Ca2+) function as ubiquitous messengers that play an essential role in signal transduction and control a wide array of cellular functions. In T cells, the signal transduction pathways triggered by the activation of the T-cell receptor (TCR)/CD3 complex lead to the immediate activation of transcription factors that regulate a variety of activation-associated genes. Many of them are cytokines and surface receptors that play an important role in coordinating the immune response (Crabtree and Clipstone, 1994). The signal transduction pathways involved in T-cell activation are initiated by the activation of phospholipase C
by specific tyrosine kinases at the lipid rafts, resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate and generation of inositol-(1,4,5)-triphosphate (InsP3) and diacylglycerol. InsP3 binds to the InsP3 receptor (InsP3R) in the membrane of the ER, which is the main intracellular Ca2+ store, and initiates release of the stored Ca2+. Depletion of the ER of Ca2+ activates store-operated calcium release-activated Ca2+ (CRAC) channels in the plasma membrane that is an essential step during T-lymphocyte activation (Lewis, 2001). The Ca2+ influx operated through these channels is critical to induce an effective immune response (Feske et al., 2001).
Nuclear factor of activated T cells (NFAT) is a family of transcription factors present in cells and tissues both inside and outside of the immune system and is composed of at least four structurally related members, NFAT1, NFAT2, NFAT3, and NFAT4, that are expressed in the cytoplasm of the resting cells as well as the constitutively nuclear NFAT5 member (Hogan et al., 2003). As a consequence of an increase of [Ca2+]i levels, calcineurin is activated. This Ca2+-calmodulin-dependent protein phosphatase subsequently dephosphorylates the NFAT, triggering its nuclear shuttling. Once in the nucleus, NFAT binds to the DNA either alone or in conjunction with other transcriptional partners (Macian, 2005). The activator protein 1 (AP-1) is considered the major interacting partner of NFAT and is also controlled by TCR-dependent Ca2+ signals through Ca2+-dependent kinases (Rao et al., 1997). NFAT was first described as an inducible regulatory complex critical for transcriptional induction of IL-2 gene in activated T cells (Shaw et al., 1988) but was subsequently shown to regulate the transcription of many other cytokines and T-cell activation-induced proteins (Macian, 2005).
The transcription factor nuclear factor-B (NF-B) is one of the key regulators of genes involved in the immune/inflammatory response as well as in survival from apoptosis. NF-B is an inducible transcription factor that interacts with a family of inhibitory IB proteins, of which IB is the best characterized (Karin and Ben-Neriah, 2000). In most cell types, these proteins sequester NF-B in the cytoplasm by masking its nuclear localization sequence. Antigen stimulation in T cells triggers a signaling pathway that results in the phosphorylation, ubiquitination, and subsequent degradation of IB proteins, with the eventual translocation of NF-B from the cytoplasm to the nucleus (Karin and Ben-Neriah, 2000). Although the signaling pathways leading to NF-B activation downstream to TCR engagement are not fully understood, it has been shown that NF-B activity is also influenced by a rise in [Ca2+]i (Feng et al., 2002).
Since basiliolides are structurally unrelated to TG, it was interesting to further investigate their biological profile and compare it with that of TG. We now report that basiliolides mobilize Ca2+ from the ER with a mechanism apparently different from that of TG, which qualifies these compounds as new probes to investigate intracellular calcium homeostasis.
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B mAb was a gift from R. T. Hay (Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Scotland), and the rabbit polyclonal anti-NFAT1 was a gift from J. M. Redondo (Centro de Bioquimica y Biologia Molecular, Madrid, Spain). The anti-phospho-ERK1 + 2 (sc-7383) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the mAbs anti-phospho-JNK (9255S) and anti-phospho-p65 (3031S) were from New England Biolabs (Hitchin, UK). Thapsigargin and the tetracyclic C19 dilactones basiliolide A1, B, and C were isolated from Thapsia garganica as described previously (Appendino et al., 2005). BTP-2 was from Calbiochem (San Diego, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO).
Measurement of TNF- Synthesis. Jurkat cells (106/ml) were treated as indicated for 6 h in complete medium. After culture, supernatants were harvested and centrifuged for 10 min at 10,000g, and the levels of TNF- in the supernatant were measured by enzyme-linked immunosorbent assay (Immunotools, Friesoythe, Germany) according to the manufacturer's instructions. Experiments were carried out in triplicate. Analysis was performed using analysis of variance followed by the Student-Newman-Keuls method with values of p < 0.05 considered to be significant.
Cell Cycle Analysis and Cytotoxicity Assays. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. In brief, cells were collected after treatments, washed twice with phosphate-buffered saline (PBS), and fixed with 70% ethanol for 24 h at 4°C. The cells were then washed twice with PBS solution and subjected to RNA digestion (50 U/ml RNase-A) and 20 µg/ml propidium iodide staining in PBS for 1 h at room temperature. The cells were analyzed by cytofluorimetry. Under these conditions, low-molecular-weight DNA leaks from the ethanol-fixed cells, and the subsequent staining allows the determination of the percentage of apoptotic cells (sub-G0/G1 fraction). For cytotoxicity analysis, Jurkat cells were seeded in 96-well plates in complete medium and treated with increasing doses of either thapsigargin or basiliolide A1 for the indicated times. Samples were then diluted with 300 µl of PBS and incubated for 1 min at room temperature in the presence of 10 µg/ml propidium iodide. After incubation, cells were immediately analyzed by flow cytometry.
Ca2+ Mobilization Assay in Jurkat Cells. Jurkat cells were incubated for 1 h at 37°C in Tyrode's salt solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 0.4 mM NaH2PO4, 12.0 mM NaHCO3, and 5.6 mM D-glucose) containing 5 µM Indo1-AM (Invitrogen) for 30 min at 37°C in the dark. Cells were then harvested, washed three times with buffer to remove extracellular Indo1 dye, readjusted to 106 cells/ml in the appropriate buffer, and analyzed in a spectrofluorimeter operated in the ratio mode (model F-2500; Hitachi Ltd., Tokyo, Japan) under continuous stirring and at a constant temperature of 37°C using a water-jacketed device. After 5-min accommodation to equilibrate temperatures, samples were excited at 338 nm, and emission was collected at 405 and 485 nm, corresponding to the fluorescence emitted by Ca2+-bound and -free Indo1, respectively. [Ca2+]i was calculated using the ratio values between bound and free Indo1 fluorescence and assuming an Indo1 Kd for Ca2+ of 0.23 µM. Maximal and minimal ratio values for calculations were determined by the addition at the end of the measurements of 10 µM ionomycin or 4 mM EGTA, respectively. [Ca2+]i changes are presented as changes in the ratio of bound to free calcium (340 nm/380 nm). To determine the rate of Ca2+ entry, Indo1-loaded cells were suspended in nominally Ca2+-free buffer (50 mM HEPES, pH 7.5, and 200 mM NaCl) and stimulated with basiliolide A as indicated. Then, 1 mM CaCl2 was introduced in the medium, and the ensuing increase in [Ca2+]i was monitored.
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). The background obtained with the lysis buffer was subtracted in each experimental value, and the specific transactivation was expressed as total relative light unit (RLU) induction. All the experiments were repeated at least three times. Analysis was performed using analysis of variance followed by the Student-Newman-Keuls method with values of p < 0.05 considered to be significant. The AP-1-Luc, NFAT-Luc, KBF-Luc, IL-2-Luc, and TNF-Luc plasmids have been described previously (Sancho et al., 2004). Western Blots. Jurkat cells (106 cells/ml) were stimulated as indicated and then washed with PBS and resuspended in lysis buffer (20 mM HEPES, pH 8.0, 0.35 M NaCl, 0.1 mM EGTA, 0.5 mM EDTA, 1 mM MgCl2, 20% glycerol, 1 mM dithiothreitol, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, 0.5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) containing 0.5% Nonidet P-40. Cells were incubated for 15 min at 4°C, and cellular proteins were obtained by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA), and 30 µg of proteins was boiled in Laemmli buffer and electrophoresed in 10% SDS-polyacrylamide gels or in 6% SDS-polyacrylamide gels (for NFAT detection). Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V; 4°C) for 1 h. Blots were blocked in Tris-buffered saline solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C, and immunodetection of specific proteins was carried out with primary antibodies using an ECL system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
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). To study the effects of these novel compounds in mammalian cells, the [Ca2+]i in Indo1-loaded Jurkat cells was analyzed. Addition of increasing concentrations of basiliolide A1 to the cells resulted in a rapid increase in [Ca2+]i comparable with the increase induced by TG (Fig. 1, A and B). The Ca2+ increase was maintained throughout the recording in response to both compounds, although TG was slightly more potent than basiliolide A1 (Fig. 1, A and B). When ER intracellular stores were depleted by thapsigargin, basiliolide A1 was no longer able to mobilize [Ca2+]i (Fig. 1C), but TG could still mobilize [Ca2+]i in basiliolide A1-treated cells (Fig. 1D). These observations suggest that thapsigargin and basiliolide A1 target the Ca2+ ER stores through different mechanisms.
The rapid and sustained effect of basiliolide A on [Ca2+]i mobilization suggests the participation of CRAC in such mobilization. To dissect the biochemical mechanism for this basiliolide A-induced calcium mobilization, we examined the effects of EGTA, an extracellular calcium chelator, and BAPTA-AM, a compound that can enter cells in an ester form, but that is then hydrolyzed by intracellular esterases and retained in the cytoplasm, acting as a specific calcium chelator. A different pattern of basililoide A-induced calcium mobilization was observed in the presence of either EGTA or BAPTA-AM (Fig. 2A). When cells were treated with BAPTA-AM, the early elevation of intracellular calcium disappeared, but a slow calcium accumulation persisted. On the contrary, cells treated with EGTA, which cannot enter the cell, showed the rapid and early phase of calcium mobilization in response to basiliolide A, but it disappeared within seconds until it reached the basal levels. These data indicate that basiliolide A induces calcium mobilization by at least two coupled mechanisms. The first mechanism would be mediated by the release of calcium from intracellular stores (inhibited by BAPTA-AM), and the second mechanism would be mediated by the entry of extracellular calcium, inhibited by EGTA and probably induced by the opening of cell surface calcium channels. The effects of BAPTA as calcium chelator may be sufficient to quench a limited amount of the calcium release to the cytosol (internal stores), but it is not able to quench completely the high [Ca2+]i induced by basiliolide A in Jurkat cells. Next, we reasoned that basiliolide A1-induced Ca2+ depletion of the ER should activate CRAC channels in the plasma membrane and to address this point, we preincubated the cells for 24 h with the potent CRAC channel inhibitor BTP-2 (Zitt et al., 2004). We found that basiliolide A1 induced the early phase of calcium mobilization, but the late phase was completely prevented by BTP-2 (Fig. 2A). To further confirm the activity of basiliolide A1 on calcium mobilization from ER stores and CRAC channels, we performed experiments in nominally free calcium, and we observed that basiliolide A induces an immediate [Ca2+]i mobilization that was further enhanced by the addition of CaCl2 to the cuvette (Fig. 2B). As expected, the [Ca2+]i increase mediated by the addition of CaCl2 was almost completely inhibited by preincubation of the cells with BTP-2 (Fig. 2C), but not by the L-type calcium channels blockers verapamil and nifedipine (data not shown).
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; Kaneko and Tsukamoto, 1994). Since basiliolide A also depletes ER intracellular [Ca2+]i stores in Jurkat cells, we investigated its potential proapoptotic activity. The cells were incubated with 5 µM basiliolide A, 1 µM thapsigargin, or the calcium ionophore ionomycin (1 µM) for 18 h, and the hypodiploidy (i.e., loss of fragmented DNA) was detected, using propidium iodide staining, as a marker for apoptosis. Figure 3A shows that both thapsigargin and ionomycin were able to induce a clear increase in the percentage of hypodiploid cells (57.9 and 23.5%, respectively). In contrast, basiliolide A1 lacked apoptotic activity, indicating that this compound does not induce the ER stress required to activate the apoptotic program (Lang et al., 2005). The rise of [Ca2+]i is also involved in the necrotic pathway of cell death that cannot be measured by cell cycle analysis; therefore, we sought to study the effects of basiliolide A1 and TG on cellular toxicity that was evaluated by propidium iodide staining and flow cytometry. Jurkat cells were treated with increasing doses of both compounds, and cell viability was tested after 6, 12, and 18 h. As depicted in Fig. 3B, basiliolide A1 lacked cytotoxic effects at the concentrations and times analyzed. In contrast, TG clearly induced cell death with all the concentration tested.
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B and AP-1 Activation. Whereas [Ca2+]i is clearly involved in NFAT dephosphorylation and nuclear translocation, its effect on NF-B and AP-1 activation is more indirect (Rao et al., 1997). However, ER stress induced by thapsigargin has been shown to activate the NF-B and mitogen-activated protein kinase signaling pathway (Maloney et al., 1999; Rosado and Sage, 2001; Engedal et al., 2002; Leonardi et al., 2002). Thus, we investigated some of major biochemical pathways activated by basiliolide A leading to NF-B and AP-1 activation in Jurkat cells. We found that basiliolide A1 alone did not induce IB degradation nor RelA (NF-B subunit) phosphorylation (ser536), but it synergized with PMA to induce a complete degradation of this NF-B inhibitory protein (Fig. 5A). We also detected that basiliolide A1 and ionomycin increased PMA-mediated JNK1 and JNK2 phosphorylation, whereas PMA-induced ERK1 and ERK2 phosphorylation was not affected (Fig. 5A). Accordingly, basiliolide A1 synergized with PMA to induce NF-B- and AP-1-dependent transcriptional activation (Fig. 5, B and C).
IL-2 and TNF- gene promoter are regulated by the coordinated action of NFAT, NF-B, and AP-1 transcription factors that are activated by antigen receptor engagement plus an accessory signal usually supplied by the antigen-presenting cell (Crabtree and Clipstone, 1994). Agents that bypass these receptors, such as PMA and ionomycin, can mimic T-cell activation in the Jurkat cells. Thus, the costimulatory effect of basiliolide A1 was studied by transfecting Jurkat cells with the reporter plasmids IL-2-Luc and TNF--Luc. After transfection, cells were treated with PMA alone or in combination with basiliolide A1 or ionomycin as a control for 6 h and tested for luciferase activity. In Fig. 6, it is shown that basiliolide A1 is a potent coactivator with PMA to induce the luciferase expression driven by both calcium-dependent cellular gene promoters (Fig. 6, A and B). Interestingly, basiliolide A1 was more effective than ionomycin to induce TNF- release from the cells (Fig. 6C), suggesting that basiliolide A1 may control the release of cytokines at both transcriptional and post-transcriptional levels.
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The Position 15 of Basiliolide Structure Is Critical for NFAT Activation in Jurkat Cells. To gain insight on the structure-activity relationships of basiliolides, the activity of basiliolide A1 was compared with that of its two more oxidized analogs, basiliolides B and C (Fig. 7). Although basiliolide B, differing in the oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. In contrast to basiliolide A, high concentrations of thapsigargin were less effective than lower concentrations to induce NFAT-dependent luciferase activity.
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Under physiological conditions, Ca2+ release from ER to cytosol can be induced in T cells by the direct binding of the second messengers InsP3 and cyclic adenosine diphosphoribose via the InsP3R and the ryanodine receptor (RyR), respectively (Quintana et al., 2005). Basiliolide A1 might interfere with upstream mechanisms that lead to the generation of some of these second messengers. However, basiliolide A1 could induce calcium mobilization both in the parental Jurkat clone and in a phospholipase C1-defficient line (Irvin et al., 2000; data not shown), whereas the RyR antagonist dantrolene was unable to prevent both [Ca2+]i elevation and NFAT activation induced by basiliolide. These observations rule out a direct activation of these ER receptors by basiliolide A1, suggesting an indirect interaction with them. Notwithstanding, at present we cannot discard the possibility that basiliolide A interacts directly either with InsP3R or RyR. It has been shown recently that Homer, a scaffold protein, physically associates with the ryanodine receptors type 1, regulating gating responses to Ca2+ and caffeine (Feng et al., 2002) as well as NFAT-dependent signaling (Stiber et al., 2005). It is therefore not inconceivable that, just like caffeine, basiliolide A1 also modulates RyR in a Homer-dependent manner (Feng et al., 2002).
SERCA is a critical enzyme that pumps Ca2+ from the cytosol into the ER lumen to maintain a low [Ca2+]i. The sustained inhibition of this activity causes depletion of intracellular Ca2+ stores and activation of capacitative Ca2+ entry, generating supramolecular [Ca2+]i in the cytosol. In Jurkat and related cell types, this activates apoptotic pathways (Jayaraman and Marks, 1997). The very tight binding of thapsigargin to all currently known SERCAs causes an irreversible inhibition that persists after removal of the excess inhibitor (Waldron et al., 1994), an observation that can explain, or contribute to, the potent apoptotic activity of this compound. Conversely, basiliolide A1 was unable to induce apoptosis in Jurkat cells, despite its alleged SERCA inhibitory activity (Appendino et al., 2005). It is therefore tempting to suggest that basiliolide A1 acts as a reversible SERCA inhibitor, not unlike 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a SERCA blocker that protects HeLa cells from ceramide-induced apoptosis (Pinton et al., 2001). The reversible activity of basiliolide A1 is supported by the observation that Jurkat cells treated with this compound for 1 h and then washed and cultured again for 12 h in calcium-containing medium were still sensible to the [Ca2+]-mobilizing properties of basiliolide A1 (data not shown). To conciliate these observations, we can assume that the reduction of the ER [Ca2+]i by basiliolide A1 leads to a constant release of Ca2+ to the cytosol and reuptake by the ER, whose long kinetics makes it possible for calcium to equilibrate between different intracellular organelles. This calcium leakage can be translated into calcium-dependent gene transcription and other cellular functions but not into the induction of cell death. Thapsigargin and ionomycin generate instead very high [Ca2+]i and quickly induce apoptosis. We are currently investigating whether basiliolide A1 can protect cells from ceramide-induced apoptosis in T cells and in other cell types. Another interesting difference between basiliolide A1 and thapsigargin is that NFAT activation by thapsigargin has a complex kinetics, with high concentrations being less efficient than lower concentrations. This could be explained in part by an increase in apoptosis, since a higher percentage of cell death was found in Jurkat cells treated with 5 µM TG compared with cells treated with lower concentrations of this SERCA inhibitor. Interestingly, it has been proposed that high concentrations of thapsigargin can also have inhibitory effect on Ca2+ or Mn2+ entry from the medium (Mason et al., 1991). Taken together, these observations qualify basiliolides as a novel class of molecular probes to study calcium homeostasis, characterized by lack of apoptotic and CRAC inhibitory activity.
NFAT, NF-B, and AP-1 are probably the three most important transcription factor families in T cells, all of them being activated downstream from TCR engagement in a Ca2+-dependent manner. Whereas [Ca2+]i is clearly involved in activation of NFAT, the role of [Ca2+]i may be regarded as being more indirect for NF-B and AP-1 (Feske et al., 2001; Li and Verma, 2002). Accordingly, we found that basiliolide A1 is potent costimulator of the NF-B and the AP-1 pathways in T cells, but this activity was not restricted to T cells, since we observed that basiliolides also regulates NFAT and NF-B activity in neuronal cells (our unpublished data). This is of special relevance, since both transcription factors can protect neurons from cell death both "in vivo" and "in vitro" (Fridmacher et al., 2003; Benedito et al., 2005). Noncytotoxic compounds that mobilize calcium by targeting the ER are currently of great interest for the treatment of neurodegenerative diseases
The differences between the activity of basiliolides A1, B, and C demonstrate the existence of definite structure-activity relationships within these compounds and should spur activity aimed at the isolation of further members of this class of compounds and/or to their semisynthetic modification. As a final observation, it is interesting to remark that T. garganica is one of the oldest medicinal plants, and it is therefore surprising that this treasure trove of bioactive compounds was overlooked for so long in terms of phytochemical and pharmacological investigations.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: TG, thapsigargin; SERCA, sarco(endo)plasmic reticulum Ca2+ ATPase; ER, endoplasmic reticulum; TCR, T-cell receptor; InsP3, inositol-(1,4,5)-triphosphate; InsP3R, inositol-(1,4,5)-triphosphate receptor; CRAC, calcium release-activated Ca2+ channel; NFAT, nuclear factor of activated T cells; AP-1, activator protein-1; IL, interleukin; NF-B, nuclear factor-B; mAb, monoclonal antibody; ERK, extracellular regulated kinase; JNK, c-Jun NH2-terminal kinase; BTP-2, N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; AM, acetoxymethyl ester, RLU, relative light unit; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PMA, phorbol 12-myristate 13-acetate; RyR, ryanodine receptor; BSD, basiliolide.
Address correspondence to: Dr. Eduardo Muñoz, Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Medicina, Avda. de Menéndez Pidal s/n, Universidad de Córdoba, 14004 Córdoba, Spain. E-mail: fi1muble{at}uco.es
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