thapsigargin and jasplakinolide

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca(2+) concentration ([Ca(2+)](i)), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca(2+)](i), as measured by fura-2 fluorescence, was transiently increased in a strain amplitude-dependent manner. The elevation of [Ca(2+)](i) induced by stretch was not evident in the Ca(2+)-free solution and was blocked by Gd(3+), a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca(2+)](i). In contrast, increases in [Ca(2+)](i) induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca(2+)](i). A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca(2+) channels. In conclusion, mechanical stretch activates Ca(2+) influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca(2+) influx pathways such as receptor-operated and store-operated Ca(2+) entries in HPMVECs. These results suggest that abnormal Ca(2+) homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.

Topics: Actins; Calcium; Cells, Cultured; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Lung; Lysophospholipids; Microcirculation; Microscopy, Fluorescence; Models, Chemical; Sphingosine; Stress, Mechanical; Thapsigargin

Bax inhibitor 1 (BI-1), a transmembrane protein with Ca2+ channel-like activity, has antiapoptotic and anticancer activities. Cells overexpressing BI-1 demonstrated increased cell adhesion. Using a proteomics tool, we found that BI-1 interacted with gamma-actin via leucines 221 and 225 and could control actin polymerization and cell adhesion. Among BI-1-/- cells and cells transfected with BI-1 small interfering RNA (siRNA), levels of actin polymerization and cell adhesion were lower than those among BI-1+/+ cells and cells transfected with nonspecific siRNA. BI-1 acts as a leaky Ca2+ channel, but mutations of the actin binding sites (L221A, L225A, and L221A/L225A) did not change intra-endoplasmic reticulum Ca2+, although deleting the C-terminal motif (EKDKKKEKK) did. However, store-operated Ca2+ entry (SOCE) is activated in cells expressing BI-1 but not in cells expressing actin binding site mutants, even those with the intact C-terminal motif. Consistently, actin polymerization and cell adhesion were inhibited among all the mutant cells. Compared to BI-1+/+ cells, BI-1-/- cells inhibited SOCE, actin polymerization, and cell adhesion. Endogenous BI-1 knockdown cells showed a similar pattern. The C-terminal peptide of BI-1 (LMMLILAMNRKDKKKEKK) polymerized actin even after the deletion of four or six charged C-terminal residues. This indicates that the actin binding site containing L221 to D231 of BI-1 is responsible for actin interaction and that the C-terminal motif has only a supporting role. The intact C-terminal peptide also bundled actin and increased cell adhesion. The results of experiments with whole recombinant BI-1 reconstituted in membranes also coincide well with the results obtained with peptides. In summary, BI-1 increased actin polymerization and cell adhesion through Ca2+ regulation and actin interaction.

Topics: Actins; Amino Acid Sequence; Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Binding Sites; Calcium; Cell Adhesion; Cell Line; Depsipeptides; Enzyme Inhibitors; Humans; Membrane Proteins; Mice; Mice, Knockout; Molecular Sequence Data; Patch-Clamp Techniques; Peptides; Protein Binding; RNA, Small Interfering; Thapsigargin

Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia.. Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4-5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2.. Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia.. Our results suggest that spermatozoa from asthenozoospermic patients present a reduced responsiveness to progesterone.

Topics: Actin Cytoskeleton; Asthenozoospermia; Biological Transport; Calcium; Calcium Signaling; Cytochalasin D; Cytosol; Depsipeptides; Enzyme Inhibitors; Humans; Male; Progesterone; Progestins; Spermatozoa; Thapsigargin

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.

Topics: Actins; Benzyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Manganese; Potassium; Sodium; Sodium-Calcium Exchanger; Thapsigargin; Thiazolidines; Thiourea

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.

Topics: Animals; Benzimidazoles; Calcium Signaling; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cholecystokinin; Cytochalasin D; Cytoskeleton; Depsipeptides; Dose-Response Relationship, Drug; Hydrogen Peroxide; Indoles; Intracellular Fluid; Male; Maleimides; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oligomycins; Organotin Compounds; Pancreas, Exocrine; Protein Kinase C; Ruthenium Compounds; Salicylates; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sincalide; Thapsigargin

Numerous studies have demonstrated that members of the transient receptor potential (TRP) superfamily of channels are involved in regulated Ca2+ entry. Additionally, most Ca2+-permeable channels are themselves regulated by Ca2+, often in complex ways. In the current study, we have investigated the regulation of TRPC7, a channel known to be potentially activated by both store-operated mechanisms and non-store-operated mechanisms involving diacylglycerols. Surprisingly, we found that activation of TRPC7 channels by diacylglycerol was blocked by the SERCA pump inhibitor thapsigargin. The structurally related channel, TRPC3, was similarly inhibited. This effect depended on extracellular calcium and on the driving force for Ca2+ entry. The inhibition is not due to calcium entry through store-operated channels but rather results from calcium entry through TRPC7 channels themselves. The effect of thapsigargin was prevented by inhibition of calmodulin and was mimicked by pharmacological disruption of the actin cytoskeleton. Our results suggest the presence of a novel mechanism involving negative regulation of TRPC channels by calcium entering through the channels. Under physiological conditions, this negative feedback by calcium is attenuated by the presence of closely associated SERCA pumps.

Topics: Adenosine Triphosphate; Boron Compounds; Calcium; Calcium-Transporting ATPases; Calmodulin; Cations; Cytochalasin B; Cytoskeleton; Depsipeptides; Diglycerides; Gadolinium; Humans; Imidazoles; Indoles; Ion Transport; Thapsigargin; TRPC Cation Channels

Store-mediated Ca2+ entry is thought as the main pathway for Ca2+ influx in non-excitable cells. Although a role for the actin cytoskeleton in store-mediated Ca2+ entry has been proposed in some cell types, the role of actin cytoskeleton in store-mediated Ca2+ entry is still a controversy. To address this question, the effects of cytoskeletal modifiers on store-mediated Ca2+ entry in pleural mesothelial cells were examined. Thapsigargin (1 microM) induced a sufficient signal for the activation of store-mediated Ca2+ entry in pleural mesothelial cells. In the absence of extracellular Ca2+, thapsigargin induced only a transient elevation of [Ca2+]i. Moreover, re-addition of Ca2+ increased the elevation of [Ca2+]i. Passive elevations in [Ca2+]i without thapsigargin, which is induced from Ca2+ containing solution switch to Ca2+ free solution and re-add Ca2+ containing solution, were not observed in pleural mesothelial cells. Thapsigargin-induced Ca2+ entry was still present after nifedipine (1 microM) treatment. However, SKF96365 (1 microM) blocked thapsigargin-induced Ca2+ entry. Mycalolide B (1 microM) completely disrupts actin cytoskeleton in pleural mesothelial cells, but thapsigargin-induced store-mediated Ca2+ entry was preserved. Jasplakinolide (3 microM) prevented thapsigargin-induced store-mediated Ca2+ entry. These results suggest that store-mediated Ca2+ entry in pleural mesothelial cells may be mediated by a recently proposed secretion-like coupling model for store-mediated Ca2+ entry.

Topics: Actins; Animals; Calcium; Cell Shape; Cells, Cultured; Cytochalasin D; Cytoskeleton; Depsipeptides; Epithelial Cells; Imidazoles; Marine Toxins; Microscopy, Confocal; Nifedipine; Oxazoles; Pleura; Rats; Thapsigargin

Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60(src), which is independent on Ca2+ entry. pp60(src) activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.

Topics: Actins; Animals; Biopolymers; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytochalasin D; Cytoskeleton; Depsipeptides; Ion Transport; Male; Mice; Pancreas; Peptides, Cyclic; Proto-Oncogene Proteins pp60(c-src); Spectrometry, Fluorescence; Thapsigargin; Thiazoles; Thiazolidines

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.

Topics: Actins; Analysis of Variance; Animals; Calcium Signaling; Cations, Divalent; Cell Degranulation; Cross-Linking Reagents; Cytochalasin D; Depsipeptides; Dinitrophenols; Humans; Immunoglobulin E; Inositol 1,4,5-Trisphosphate; Macrocyclic Compounds; Mast Cells; Oxazoles; Peptides, Cyclic; Rats; Receptors, IgE; Serum Albumin; Thapsigargin; Tumor Cells, Cultured

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells

Topics: Animals; Basophils; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Cell Size; Cytochalasin D; Cytoskeleton; Depsipeptides; Enzyme Inhibitors; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lithium; Marine Toxins; Microscopy, Fluorescence; Nocodazole; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Rats; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Time Factors

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2, 5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)-CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.

Topics: Calcium; Calpain; Cell Adhesion; Cells, Cultured; Cytoskeleton; Depsipeptides; Humans; Hydroquinones; Intercellular Adhesion Molecule-1; Ionomycin; Lymphocyte Function-Associated Antigen-1; Microscopy, Confocal; Peptides, Cyclic; Signal Transduction; T-Lymphocytes; Thapsigargin