thapsigargin and 2-5-di-tert-butylhydroquinone

This review is derived from the symposium held at the 66th Annual Meeting of the Japanese Pharmacological Society (March, 1993). The symposium consisted of six invited papers whose general theme was the application of recently found ATPase inhibitors selective to SR- and ER-Ca(2+)-ATPase to the analyses of the physiological and pharmacological roles of endoplasmic and sarcoplasmic reticulum Ca stores. Inhibitors used were: thapsigargin, cyclopiazonic acid, 2,5-di-(t-butyl)-1,4-benzohydroquinone and 3',3",5',5"-tetraiodosulfophthalein. Gingerol was found to facilitate the action of the ATPase. In either smooth, cardiac or skeletal muscle, sympathetic neurons or several cell lines these inhibitors affected a variety of cell functions and conditions such as contraction, ionic conductance and excitability of the plasma membrane, regulation of intracellular free Ca2+ concentration, transport of viral glycoprotein to the cell surface. Many of these studies utilized either single or cultured cell preparations or skinned muscle. These inhibitors were shown to be useful tools for investigating the SR and ER functioning as Ca sources or Ca sequestrating pumps, and further for estimating the contribution of ER or SR to regulating the flux of Ca2+ and other ions through the plasma membrane. Results of analyses using these inhibitors are discussed.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Membrane; Drug Design; Endoplasmic Reticulum; Hydroquinones; Indoles; Sarcoplasmic Reticulum; Terpenes; Thapsigargin

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca

Topics: Animals; Boron Compounds; Calcium; Calcium Chelating Agents; Computational Biology; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Gene Expression; Homeostasis; Hydroquinones; Intracellular Calcium-Sensing Proteins; Manganese; Protozoan Proteins; Thapsigargin; Transient Receptor Potential Channels; Trypanosoma; Trypanosomiasis

The antidepressant, sertraline, has been shown to have diverse in vitro effects. This study examined whether sertraline altered [Ca(2+)](i) in MG63 human osteosarcoma cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. At 50-200 µM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent manner. Ca(2+) response was decreased by removing extracellular Ca(2+), suggesting that Ca(2+) entry and release contributed to the [Ca(2+)](i) signal. Sertraline-induced Ca(2+) entry was inhibited by nifedipine, La(3+), Gd(3+), and SK&F96365. When extracellular Ca(2+) was removed, pretreatment with the endoplasmic reticulum (ER) Ca(2+) pump inhibitor, thapsigargin, or 2,5-di-tert-butylhydroquinone (BHQ) abolished the sertraline-evoked [Ca(2+)](i) rise. Incubation with sertraline also abolished the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished the sertraline-induced [Ca(2+)](i) rise. At 20-30 µM, overnight treatment with sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data demonstrate that sertraline (30 µM) evoked apoptosis. Sertraline (20 and 30 µM) also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, sertraline evoked a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the ER and Ca(2+) entry by L-type Ca(2+) channels and store-operated Ca(2+) channels. Sertraline induced cell death that may involve apoptosis by mitochondrial pathways.

Topics: Apoptosis; Calcium; Calcium Channels, L-Type; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Estrenes; Humans; Hydroquinones; Mitochondria; Osteosarcoma; Pyrrolidinones; Reactive Oxygen Species; Selective Serotonin Reuptake Inhibitors; Sertraline; Thapsigargin; Type C Phospholipases

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+) ](i) ) in MDCK renal tubular cells using the Ca(2+) -sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+) ](i) in a concentration-dependent manner. The signal was reduced by 80% by removing extracellular Ca(2+) . Methoxychlor-induced Ca(2+) entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited methoxychlor-induced [Ca(2+) ](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+) ](i) rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor-induced [Ca(2+) ](i) rise. At 5-15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca(2+) ](i) rise by inducing phospholipase C-dependent Ca(2+) release from multiple stores and Ca(2+) entry via protein kinase C- and econazole-sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration-dependent, Ca(2+) -independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.

Topics: Animals; Annexin A5; Apoptosis; Calcium; Calcium Signaling; Cell Movement; Cell Survival; Cytosol; Dogs; Econazole; Egtazic Acid; Endoplasmic Reticulum; Estrenes; Fura-2; Hydroquinones; Madin Darby Canine Kidney Cells; Methoxychlor; Nifedipine; Protein Kinase C; Pyrrolidinones; Reactive Oxygen Species; Thapsigargin; Type C Phospholipases

The exact cellular target for the potent anti-cancer agent hypericin has not yet been determined; this thus encourages the application of computational chemistry tools to be employed in order to provide insights that can be employed in further drug development studies. In the present study computational docking and molecular dynamics simulations are applied to investigate possible interactions between hypericin and the Ca(2+) pump SERCA as proposed in the literature. Hypericin was found to bind strongly both in pockets within the transmembrane region and in the cytosolic region of the protein, although the two studied isoforms of SERCA differ slightly in their preferred binding sites. The calculated binding energies for hypericin in the four investigated sites were of the same magnitude as for thapsigargin (TG), the most potent SERCA inhibitor, or in the range between TG and di-tert-butylhydroquinone (BHQ), which is also known to possess inhibitory activity. The hydrophobic character of hypericin indicates that the molecule initially binds in the ER membrane from which it diffuses into the transmembrane region of the protein and to binding pockets therein. The transmembrane TG and BHQ binding pockets provide suitable locations for hypericin as they allow for favourable interactions with the lipid tails that surround these. High binding energies were noted for hypericin in these pockets and are expected to constitute highly possible binding sites due to their accessibility from the ER membrane. Hypericin most likely binds to both isoforms of SERCA and acts as an inhibitor or, under light irradiation, as a singlet oxygen generator that in turn degrades the protein or induces lipid peroxidation.

Topics: Anthracenes; Antineoplastic Agents; Enzyme Inhibitors; Humans; Hydroquinones; Models, Molecular; Molecular Dynamics Simulation; Molecular Structure; Perylene; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Structure-Activity Relationship; Thapsigargin

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 1-50 μM evoked a [Ca(2+)](i) rise in a concentration-dependent manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 10-75 μM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 μM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.

Topics: Apoptosis; Calcium; Calcium Channel Blockers; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Econazole; Endoplasmic Reticulum; Fura-2; Humans; Hydroquinones; Imidazoles; Indoles; Liver Neoplasms; Nifedipine; Phospholipases A2; Protein Kinase C; Tetrazolium Salts; Thapsigargin

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 microM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 microM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.

Topics: Blood Platelets; Blotting, Western; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Membrane; Cells, Cultured; Electroporation; Enzyme Inhibitors; Flufenamic Acid; Humans; Hydroquinones; Immunoprecipitation; Inositol 1,4,5-Trisphosphate Receptors; Microtubules; Protein Binding; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; TRPC Cation Channels; TRPC6 Cation Channel

The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca(2+) ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca(2+) binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca(2+) in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca(2+). We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca(2+). The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1 approximately P . 2Ca(2+) to E2-P . 2Ca(2+) transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca(2+) translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca(2+) ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.

Topics: Adenosine Triphosphate; Animals; Calcium; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors; Hydroquinones; Indoles; Isothiuronium; Protein Conformation; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Static Electricity; Thapsigargin

Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is regulated by the filling state of the intracellular Ca2+ stores. We have previously reported that a de novo conformational coupling between the type II IP3 receptor (IP3RII) and hTRPC1 channel occurs after depletion of the intracellular Ca2+ stores in human platelets, which might be involved in the activation of SOCE in these cells. Here we present for the first time direct evidence for the functional relevance of the coupling between hTRPC1 and IP3RII in SOCE in human platelets. Our data suggest that at least two pathways may contribute to SOCE in these cells. An early component, insensitive to cytochalasin D (Cyt D), is followed by a late component which is sensitive to Cyt D. Introduction of a peptide corresponding to IP3RII(317-334) (IP3BD-peptide(317-334)) in the cells by electrotransjection impairs the coupling between hTRPC1 and IP3RII but not the interaction between hTRPC1 and STIM1 induced by store depletion. Coimmunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with the IP3BD-peptide(317-334). Electrotransjection of cells with IP3BD-peptide(317-334), significantly attenuated the late stage of Ca2+ and Mn2+ entry induced by 10 nM thapsigargin (TG) or 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ), providing evidence for a functional role of the de novo coupling between hTRPC1 and IP3RII in the activation of SOCE in human platelets.

Topics: Blood Platelets; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cytochalasin D; Electroporation; Enzyme Inhibitors; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate Receptors; Manganese; Peptide Fragments; Protein Binding; Thapsigargin; Time Factors; TRPC Cation Channels

Differentiation-inducing factors (DIFs) are required for stalk cell formation in Dictyostelium discoideum. In the present study, in order to support our hypothesis that DIFs may function via increases in [Ca(2+)](c) and [H(+)](c), we investigated the combined effects of 5,5-dimethyl-2,4-oxazolidinedione (DMO, a [H(+)](c)-increasing agent), thapsigargin (Tg) and BHQ ([Ca(2+)](c)-increasing agents) on in vitro stalk cell formation in several strains. DMO, in combination with Tg or BHQ, induced stalk cell formation in a DIF-deficient mutant HM44. Although the rates of stalk cell induction by the drugs were low in the presence of cerulenin (an inhibitor of endogenous DIF production) in HM44 and V12M2 (a wild-type strain), the drugs succeeded in inducing sufficient stalk cell formation when a small amount of DIF-1 was supplied. Furthermore, co-addition of DMO, BHQ and a small amount of DIF-1 also induced sufficient stalk cell formation in AX-4 (an axenic strain) and HM1030 (dmtA(-)) but not in CT15 (dimA(-)). The drugs suppressed spore formation and promoted stalk cell formation in both HM18 (a sporogenous mutant) and 8-bromo-cAMP-stimulated V12M2. The present results suggest that DIFs function, at least in part, via increases in [Ca(2+)](c) and [H(+)](c) in D. discoideum.

Topics: Animals; Calcium; Cell Differentiation; Dictyostelium; Dimethadione; Enzyme Inhibitors; Hexanones; Hydrogen; Hydroquinones; Spores, Protozoan; Thapsigargin

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca(2+)-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-kappaB (NF-kappaB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca(2+)-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca(2+)-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC-alpha and -beta activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-kappaB-specific DNA-protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-kappaB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-kappaB activation. These data suggest that persistent inhibition of ER Ca(2+)-ATPase by TG would influence calcium release from ER Ca2+ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-kappaB activation that induces iNOS expression and NO production.

Topics: Animals; Calcimycin; Cell Line; Endoplasmic Reticulum; Enzyme Activation; Hydroquinones; Ionomycin; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Thapsigargin

Tumour necrosis factor-alpha is believed to have a deleterious role in the pathophysiology of brain injury. Tetrandrine has protective effect on neuronal cells, however, the mechanisms involved in its action have not been clearly established. The aim of this study was to investigate the role of tetrandrine on calcium-dependent tumour necrosis factor-alpha production in glia-neurone mixed cultures. Glia-neurone mixed cultures were treated by addition of Ca2+ regulating agents for a period of 6 hr. Tetrandrine or/and TMB-8 were added 30 min. before the stimulation. The supernatant tumour necrosis factor-alpha levels were quantified by enzyme-linked immunosorbent assay. Exposure of lipopolysaccharide 10 and 100 ng/ml caused significant increase in tumour necrosis factor-alpha production respectively, with no alteration in cultures treated with 1 ng/ml lipopolysaccharide. Glia-neurone mixed cultures exhibited a marked elevation in tumour necrosis factor-alpha production after exposure to CaCl2, KCl, thapsigargin, BHQ and norepinephrine in the presence of lipopolysaccharide at 1 ng/ml respectively. Tetrandrine 0.3, 1, and 3 microM concentration-dependently reduced tumour necrosis factor-alpha production evoked by CaCl2 or KCl. Tetrandrine preincubation had no significant effect on the response to Ca2+-ATPase inhibitor thapsigargin or BHQ. Norepinephrine-induced tumour necrosis factor-alpha production was significantly reduced by tetrandrine and almost abolished by combination of tetrandrine and intracellular Ca2+ release inhibitor TMB-8. These results suggested that tetrandrine at a concentration of 0.3, 1, or 3 microM inhibited tumour necrosis factor-alpha production induced by Ca2+ entry in glia-neurone mixed cultures.

Topics: Alkaloids; Animals; Benzylisoquinolines; Calcium; Calcium Channel Blockers; Calcium Chloride; Cells, Cultured; Hydroquinones; Lipopolysaccharides; Neuroglia; Neurons; Norepinephrine; Potassium Chloride; Rats; Rats, Sprague-Dawley; Thapsigargin; Tumor Necrosis Factor-alpha

Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca(2+) ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1.2Ca(2+) transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase.

Topics: Adenosine Triphosphatases; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; Blotting, Western; Calcium-Transporting ATPases; Chickens; COS Cells; DNA Mutational Analysis; DNA, Complementary; Dose-Response Relationship, Drug; Endopeptidase K; Enzyme Inhibitors; Hydroquinones; Kinetics; Lipid Bilayers; Models, Chemical; Models, Molecular; Muscle, Skeletal; Mutation; Phenylalanine; Protein Binding; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Substrate Specificity; Thapsigargin; Transfection; Transgenes

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.

Topics: Aequorin; Animals; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Carcinoma, Hepatocellular; Cytoplasm; Drug Delivery Systems; Endoplasmic Reticulum; Ethylenediamines; Fura-2; Hydroquinones; Liver; Mitochondria; Rats; Thapsigargin

Liver cells possess store-operated Ca2+ channels (SOCs) with a high selectivity for Ca2+ compared with Na+, and several types of intracellular messenger-activated non-selective cation channels with a lower selectivity for Ca2+ (NSCCs). The main role of SOCs is thought to be in refilling depleted endoplasmic reticulum Ca2+ stores [Cell Calcium 7 (1986) 1]. NSCCs may be involved in refilling intracellular stores but are also thought to have other roles in regulating the cytoplasmic-free Ca2+ and Na+ concentrations. The ability of SOCs to refill the endoplasmic reticulum Ca2+ stores in hepatocytes has not previously been compared with that of NSCCs. The aim of the present studies was to compare the ability of SOCs and maitotoxin-activated NSCCs to refill the endoplasmic reticulum in rat hepatocytes. The experiments were performed using fura-2FF and fura-2 to monitor the free Ca2+ concentrations in the endoplasmic reticulum and cytoplasmic space, respectively, a Ca2+ add-back protocol, and 2-aminoethyl diphenylborate (2-APB) to inhibit Ca2+ inflow through SOCs. In cells treated with 2,5-di-t-butylhydroquinone (DBHQ) or vasopressin to deplete the endoplasmic reticulum Ca2+ stores, then washed to remove DBHQ or vasopressin, the addition of Ca2+ caused a substantial increase in the concentration of Ca2+ in the endoplasmic reticulum and cytoplasmic space due to the activation of SOCs. These increases were inhibited 80% by 2-APB, indicating that Ca2+ inflow is predominantly through SOCs. In the presence of 2-APB (to block SOCs), maitotoxin induced a substantial increase in [Ca2+](cyt), but only a modest and slower increase in [Ca2+](er). Under these conditions, Ca2+ inflow is predominantly through maitotoxin-activated NSCCs. It is concluded that SOCs are more effective than maitotoxin-activated NSCCs in refilling the endoplasmic reticulum Ca2+ stores. The previously developed concept of a specific role for SOCs in refilling the endoplasmic reticulum is consistent with the results reported here.

Topics: Animals; Boron Compounds; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Cytoplasm; Endoplasmic Reticulum; Epinephrine; Fura-2; Hepatocytes; Hydroquinones; Inositol 1,4,5-Trisphosphate; Ion Channels; Kinetics; Male; Marine Toxins; Microscopy, Fluorescence; Oxocins; Rats; Rats, Wistar; Thapsigargin; Vasopressins

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells. Insulin evoked multiple Ca(2+) signal waveforms, 60% of which included oscillations. Although the amplitude of Ca(2+) signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca(2+)-free solutions did not affect the initiation of insulin-stimulated Ca(2+) signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA)-containing Ca(2+) stores. Insulin-stimulated Ca(2+) signals were insensitive to ryanodine. Injection of the CD38-derived Ca(2+) mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca(2+) signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca(2+)-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca(2+) signals. Insulin-stimulated Ca(2+) signals led to a Ca(2+)-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca(2+) stores in a human primary cultured cell type.

Topics: Caffeine; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Cells, Cultured; Electrophysiology; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate Receptors; Insulin; Intracellular Fluid; Islets of Langerhans; Macrocyclic Compounds; NADP; Oxazoles; Receptors, Cytoplasmic and Nuclear; Thapsigargin

Inhibiting Ca(2+) uptake by the sarcoendoplasmic reticular Ca(2+)-ATPase pump (SERCA) causes release of Ca(2+) from the endoplasmic reticulum (ER), increased cytosolic Ca(2+) ([Ca(2+)](cyt)) and depletion of ER Ca(2+) stores. These studies were designed to test the effects of SERCA inhibition on neuronal viability, using as a model the human neuroblastoma cell line, SH-SY5Y. Continuous exposure to the SERCA inhibitor thapsigargin (TG) decreased SH-SY5Y viability to <30% after 48 h exposure, and produced DNA laddering. Two other SERCA inhibitors, BHQ and cyclopiazonic acid CPA, were similarly toxic, although at 1000-fold higher concentrations. BHQ and CPA toxicity was prevented by removing drug within several hours, whereas TG toxicity was essentially irreversible. All three SERCA inhibitors caused an increase in [Ca(2+)](cyt) that was partially blocked by the ryanodine receptor inhibitors, dantrolene and DHBP. Pretreatment with 40 microM dantrolene gave substantial protection against TG- or BHQ-induced cell death but it did not inhibit death from staurosporine, which does not cause release of ER Ca(2+). DHBP (20-100 microM) also gave partial protection against TG toxicity, as did ruthenium red (2 microM), but not ryanodine (10 microM). Inhibition of capacitative Ca(2+) entry with EGTA or LaCl(3) or low extracellular Ca(2+), or chelation of [Ca(2+)](cyt) with BAPTA-AM, failed to inhibit TG toxicity, although they prevented increases in [Ca(2+)](cyt) caused by TG. Taken together, these data suggest that toxicity caused by SERCA inhibition in SH-SY5Y cells is caused by ER Ca(2+) depletion, which triggers an apparent apoptotic pathway.

Topics: Apoptosis; Brain Ischemia; Calcium; Calcium-Transporting ATPases; Cytosol; Dantrolene; Egtazic Acid; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Hydroquinones; Indoles; Muscle Relaxants, Central; Neuroblastoma; Neurons; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sesquiterpenes; Staurosporine; Thapsigargin; Tumor Cells, Cultured

In a previous study we overexpressed the thapsigargin (tg)-insensitive Pmr1 Ca(2+) pump of the Golgi apparatus of Caenorhabditis elegans in COS-1 cells and studied the properties of the Ca(2+) store into which it was integrated. Here we assessed the properties of an endogenous tg-insensitive nonmitochondrial Ca(2+) store in A7r5 and 16HBE14o- cells, which express a mammalian homologue of Pmr1. The tg-insensitive Ca(2+) store was considerably less leaky for Ca(2+) than the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA)-containing Ca(2+) store. Moreover like for the worm Pmr1 Ca(2+) pump expressed in COS-1 cells, Ca(2+) accumulation into the endogenous tg-insensitive store showed a 2 orders of magnitude lower sensitivity to cyclopiazonic acid than the SERCA-mediated transport. 2,5-Di-(tert-butyl)-1,4-benzohydroquinone was only a very weak inhibitor of the tg-insensitive Ca(2+) uptake in A7r5 and 16HBE14o- cells and in COS-1 cells overexpressing the worm Pmr1. Inositol 1,4,5-trisphosphate released 11% of the Ca(2+) accumulated in permeabilized A7r5 cells pretreated with tg with an EC(50) that was 5 times higher than for the SERCA-containing Ca(2+) store but failed to release Ca(2+) in 16HBE14o- cells. In the presence of tg, 15% of intact A7r5 cells responded to 10 microm arginine-vasopressin with a small rise in cytosolic Ca(2+) concentration after a long latency. In conclusion, A7r5 and 16HBE14o- cells express a Pmr1-containing Ca(2+) store with properties that differ substantially from the SERCA-containing Ca(2+) store.

Topics: Animals; Arginine; ATP-Binding Cassette Transporters; Bronchi; Calcium; Calcium-Transporting ATPases; Cell Line; Cells, Cultured; COS Cells; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Hydroquinones; Indoles; Inhibitory Concentration 50; Mitochondria; Muscle, Smooth; Reverse Transcriptase Polymerase Chain Reaction; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection; Vasopressins

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Topics: Animals; Binding, Competitive; Brefeldin A; Calcimycin; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Carbazoles; Cattle; Cells, Cultured; Chromaffin Cells; Dose-Response Relationship, Drug; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Hydroquinones; Indoles; Ionophores; Marine Toxins; Oxocins; Protein Subunits; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Saxitoxin; Scorpion Venoms; Sodium; Sodium Channels; Thapsigargin; Time Factors; Tritium; Veratridine

To examine whether a capacitative Ca2+ entry pathway is present in skeletal muscle, thin muscle fibre bundles were isolated from extensor digitorum longus (EDL) muscle of adult mice, and isometric tension and fura-2 signals were simultaneously measured. The sarcoplasmic reticulum (SR) in the muscle fibres was successfully depleted of Ca2+ by repetitive treatments with high-K+ solutions, initially in the absence and then in the presence of a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor. Depletion of the SR of Ca2+ enabled us for the first time to show convincingly that the vast majority of the voltage-sensitive Ca2+ store overlaps the caffeine-sensitive Ca2+ store in intact fibres from mouse EDL muscle. This conclusion was based on the observation that both high-K+ solution and caffeine failed to cause a contracture in the depleted muscle fibres. The existence of a Ca2+ influx pathway active enough to refill the depleted SR within several minutes was shown in skeletal muscle fibres. Ca2+ entry was sensitive to Ni2+, but resistant to nifedipine and was suppressed by plasma membrane depolarisation. Evidence for store-operated Ca2+ entry was provided by measurements of Mn2+ entry. Significant acceleration of Mn2+ entry was observed only when the SR was severely depleted of Ca2+. The Mn2+ influx, which was blocked by Ni2+ but not by nifedipine, was inwardly rectifying, as is the case with the Ca2+ entry. These results indicate that the store-operated Ca2+ entry is similar to the Ca2+ release-activated Ca2+ channel (CRAC) current described in other preparations.

Topics: Animals; Biological Transport; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium-Transporting ATPases; Electric Stimulation; Enzyme Inhibitors; Hydroquinones; In Vitro Techniques; Indoles; Manganese; Membrane Potentials; Mice; Muscle Fibers, Fast-Twitch; Muscle, Skeletal; Nifedipine; Potassium; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor lig

Topics: Animals; Anisoles; Arsenicals; Biological Transport; Calcium; Calcium-Transporting ATPases; Calmodulin; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Haloperidol; Hydroquinones; Kinetics; Ligands; Neurons; Ouabain; Pentazocine; Piperazines; Propylamines; Pyrrolidines; Rats; Receptors, sigma; Sigma-1 Receptor; Sodium; Sulfonamides; Tartrates; Thapsigargin; Time Factors; Tritium

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.

Topics: Animals; Calcium-Transporting ATPases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line; Cell Membrane; Clone Cells; Enzyme Inhibitors; Fura-2; Hydroquinones; Image Processing, Computer-Assisted; Ionomycin; Lithium; Meglumine; Meglumine Antimoniate; Mice; Organometallic Compounds; Organophosphates; Pituitary Gland; Potassium Chloride; Signal Transduction; Sodium-Calcium Exchanger; Tetraethylammonium; Thapsigargin

The treatment of H4-IIE cells (an immortalised liver cell line derived from the Reuber rat hepatoma) with thapsigargin, 2, 5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or pretreatment with EGTA, stimulated Ca(2+) inflow (assayed using intracellular fluo-3 and a Ca(2+) add-back protocol). No stimulation of Mn(2+) inflow by thapsigargin was detected. Thapsigargin-stimulated Ca(2+) inflow was inhibited by Gd(3+) (maximal inhibition at 2 microM Gd(3+)), the imidazole derivative SK&F 96365, and by relatively high concentrations of the voltage-operated Ca(2+) channel antagonists, verapamil, nifedipine, nicardipine and the novel dihydropyridine analogues AN406 and AN1043. The calmodulin antagonists W7, W13 and calmidazolium also inhibited thapsigargin-induced Ca(2+) inflow and release of Ca(2+) from intracellular stores. No inhibition of either Ca(2+) inflow or Ca(2+) release was observed with calmodulin antagonist KN62. Substantial inhibition of Ca(2+) inflow by calmidazolium was only observed when the inhibitor was added before thapsigargin. Pretreatment of H4-IIE cells with pertussis toxin, or treatment with brefeldin A, did not inhibit thapsigargin-stimulated Ca(2+) inflow. Compared with freshly isolated rat hepatocytes, H4-IIE cells exhibited a more diffuse actin cytoskeleton, and a more granular arrangement of the endoplasmic reticulum (ER). In contrast to freshly isolated hepatocytes, the arrangement of the ER in H4-IIE cells was not affected by pertussis toxin treatment. Western blot analysis of lysates of freshly isolated rat hepatocytes revealed two forms of G(i2(alpha)) with apparent molecular weights of 41 and 43 kDa. Analysis of H4-IIE cell lysates showed only the 41 kDa form of G(i2(alpha)) and substantially less total G(i2(alpha)) than that present in rat hepatocytes. It is concluded that H4-IIE cells possess store-operated Ca(2+) channels which do not require calmodulin for activation and exhibit properties similar to those in freshly isolated rat hepatocytes, including susceptibility to inhibition by relatively high concentrations of voltage-operated Ca(2+) channel antagonists. In contrast to rat hepatocytes, SOCs in H4-IIE cells do not require G(i2(alpha)) for activation. Possible explanations for differences in the requirement for G(i2(alpha)) in the activation of Ca(2+) inflow are briefly discussed.

Topics: Animals; Brefeldin A; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Calmodulin; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Gadolinium; Heterotrimeric GTP-Binding Proteins; Hydroquinones; Imidazoles; Indoles; Liver; Pertussis Toxin; Sulfonamides; Thapsigargin; Tumor Cells, Cultured; Virulence Factors, Bordetella

The effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ), a synthetic phenolic antioxidant and a blocker of the sarco-endoplasmic ATPase, were evaluated on low and high voltage-activated Ca(2+) currents (ICas) with rodent dorsal root ganglion, hippocampal, and motor neurons. In all cell types tested, tBHQ (IC(50) = 35 microM) blocked ICa at concentrations used to inhibit sarco-endoplasmic ATPase. This effect was specific to tBHQ because the other sarco-endoplasmic reticulum calcium ATPase pump inhibitors (thapsigargin and cyclopiazonic acid) had no effect. Selective blockade of the N-type current with omega-conotoxin GVIA and of P- (motoneuron) or Q-type currents (hippocampal neuron) with omega-agatoxin IVA indicated that tBHQ inhibited N, P, and Q types of ICa. tBHQ had no effect on nitrendipine-sensitive (L-type) and residual drug-resistant (R-type) ICa, nor on the low voltage-activated T-type ICa. Contrary to neuronal cells, the L-type ICa was inhibited by tBHQ in a differentiated mouse neuroblastoma and rat glioma hybrid cell line. Injection of cDNAs encoding the alpha1A, alpha1B, alpha1C, and alpha1E subunits into oocytes showed that tBHQ blocked ICas at the level of the pore-forming protein. This effect of tBHQ on ICa should be considered when interpreting results obtained with tBHQ used on neuronal preparations. It also may be useful for developing new strategies for the generation of more potent intracellular calcium transient inhibitors.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Calcium Channels, P-Type; Calcium Channels, Q-Type; Calcium Channels, R-Type; Calcium Channels, T-Type; Calcium-Transporting ATPases; Cells, Cultured; DNA, Complementary; Endoplasmic Reticulum; Enzyme Inhibitors; Ganglia, Spinal; Glioma; Hippocampus; Hybrid Cells; Hydroquinones; Mice; Motor Neurons; Neuroblastoma; Rats; Rats, Sprague-Dawley; Thapsigargin

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Hydroquinones; Ionomycin; Physarum polycephalum; Ryanodine Receptor Calcium Release Channel; Spectrometry, Fluorescence; Thapsigargin

C6 glioma - Ca2+ depletion - proliferation arrest morphology change - CDK inhibitor In this study, we investigated the role of the intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins in cultured C6 glioma cells. By means of microspectrofluorimetry and Ca(2+)-sensitive indicator fura-2, we found that the intracellular Ca2+ pump inhibitors, thapsigargin (TG) irreversibly and 2,5-ditert-butyl-hydroquinone (DBHQ) reversibly depleted the Ca(2+)-store accompanied with the induction of G0/G1 arrest, an increase in glial fibrillary acidic protein (GFAP) expression and morphological changes from a round flat shape to a differentiated spindle-shaped cell. The machinery underlying these changes induced by Ca(2+)-store depletion was investigated. The results indicated that Ca(2+)-store depletion caused an increased expression of p21 and p27 proteins (cyclin-dependent kinase inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: cyclin-dependent kinase 2 (CDK2), cdc2, cyclin C, cyclin D1, cyclin D3 and proliferating cell nuclear antigen (PCNA) in a time-dependent manner. These findings indicate a new function of the endoplasmic reticulum (ER) Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle-shaped morphology and induced GFAP expression were also modulated by the ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of the ER which can be manipulated by depleting the Ca2+ store.

Topics: Blotting, Western; Calcium-Transporting ATPases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cell Differentiation; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytosol; Glial Fibrillary Acidic Protein; Glioma; Humans; Hydroquinones; Microfilament Proteins; Muscle Proteins; Protein Serine-Threonine Kinases; Thapsigargin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

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

The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions.

Topics: Adenosine Triphosphate; Animals; Brain; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cytosol; Enzyme Inhibitors; Hydroquinones; Indoles; Microsomes; Phosphorylation; Swine; Synaptic Membranes; Thapsigargin; Vanadates

The affinity of sarcoplasmic reticulum Ca2+-ATPase for cyclopiazonic acid is dependent on the conformational state of the enzyme. It is high in the absence of Ca2+ but low in its presence. When Ca2+ was added to the enzyme in the presence of equimolar toxin, the apparent rate constant for Ca2+ binding was 0.6 min-1 when measured at 37 degrees C. The apparent equilibrium constant for Ca2+ dissociation increased from 0.2 to 0.6 microM at neutral pH, and from 5.9 to 37 microM at pH 6.0. The apparent equilibrium constant for Ca2+ dissociation increased progressively as the amount of toxin increased above an equimolar level. Cyclopiazonic acid decreased phosphorylation by ATP and Ca2+ when the enzyme in the absence of Ca2+ was incubated in the presence of toxin, although no effect was observed after a preliminary incubation with Ca2+ at 37 degrees C. Cyclopiazonic acid incubated with the enzyme in the presence of Ca2+ could be eliminated with a Sephadex column. However, the toxin could not be removed when it was incubated with the enzyme in the absence of Ca2+. In the latter case, cyclopiazonic acid was eliminated when the enzyme in the presence of toxin was incubated with Ca2+ at 37 degrees C. Under turnover conditions and in the presence of 10 microM ATP, the toxin-enzyme interaction can be characterized by an apparent Kd of 7 nM. With an ATP concentration of 1 mM, the enzyme was inhibited completely at a toxin/enzyme molar ratio of approximately 10. Furthermore, enzyme activity was observed to recover at a toxin/enzyme molar ratio of 1 when the Ca2+ concentration was raised, which is consistent with the competitive character of cyclopiazonic acid and Ca2+. It is concluded that ATP and Ca2+ can protect against cyclopiazonic acid inhibition.

Topics: Animals; Binding Sites; Calcium; Calcium-Transporting ATPases; Fluorescein-5-isothiocyanate; Hydroquinones; Indoles; Kinetics; Phosphorylation; Protein Binding; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Thapsigargin

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.

Topics: Aequorin; Biological Transport; Calcium Chloride; Calcium Signaling; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calreticulin; Endoplasmic Reticulum; Enzyme Inhibitors; Golgi Apparatus; Guanosine 5'-O-(3-Thiotriphosphate); HeLa Cells; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Recombinant Fusion Proteins; Ribonucleoproteins; Sialyltransferases; Strontium; Thapsigargin; Vanadates

To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells.. Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used.. Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4).. All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01).. These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.

Topics: Animals; Calcium-Transporting ATPases; Dactinomycin; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Hydroquinones; Indoles; Leukemia, Basophilic, Acute; Protein Kinase C; Rats; Tacrolimus; Thapsigargin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The present study was designed to examine the role of Ca2+ in the regulation of digestive enzyme synthesis, to determine whether changes in intracellular Ca2+ stores or cytosolic Ca2+ caused the observed effects, and to establish the steps in the pathway of protein synthesis where the regulation occurs. Protein synthesis, polysome size, and the ratio of completed to nascent polypeptides were measured as a function of Ca2+ in the intracellular stores and the cytoplasm of pancreatic acinar cells. Rat acini and rabbit pancreatic lobules were incubated in media containing 1 mM CaCl2 with the following additives: cholecystokinin (CCK) octapeptide; the inhibitors of microsomal Ca2+ ATPase, thapsigargin (THP) and 2,5-di(tertbutyl)-hydroquinone (BHQ); the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA); an inhibitor of translational initiation, 7-methylguanosine 5'-triphosphate; and an inhibitor of translation elongation, cyclohexamide. THP and BHQ depleted intracellular pools of Ca2+ and caused a sustained elevation in cytosolic [Ca2+]. Under these conditions, the polysome size diminished, and the ratio of completed proteins increased twofold relative to nascent polypeptides despite an overall decrease in net protein synthesis (55.3 +/- 2.7% of control). These effects paralleled those caused by incubation with 1 nM CCK. Incubation of pancreatic acini with BAPTA plus THP or BHQ depleted the pool [Ca2+] without changing the cytosolic [Ca2+]. In addition, these agents decreased the net protein synthesis (30.1 +/- 3.6% compared to control) and polysome size and increased the ratio of completed to nascent polypeptides to 2:1. These results suggest that depletion of intracellular stores of Ca2+ without changes in cytosolic [Ca2+] decreases protein synthesis at translational initiation.

Topics: Animals; Calcium; Calcium Chloride; Calcium-Transporting ATPases; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Hydroquinones; Kinetics; Pancreas; Polyribosomes; Protein Biosynthesis; Rabbits; Rats; Sincalide; Thapsigargin

1. Standard intracellular recording techniques with 'sharp' micropipettes were used to evoke action potentials (APs) in acutely dissociated adult nodose neurones. 2. APs induced a transient increase in [Ca2+]i (a calcium transient), recorded with fura-2, that was dependent upon [Ca2+]o and the number of APs. Over the range of one to sixty-five APs, the relation between the amplitude of the calcium transient and the number of APs was well fitted by a rectangular hyperbola (chi 2 = 3.53, r = 0.968). From one to four APs, the calcium transient-AP relation can be described by a line with a slope of 9.6 nM AP-1 (r = 0.999). 3. Charge movement corresponding to Ca2+ influx evoked by a single AP was 39 +/- 2.8 pC (mean +/- S.E.M.) and did not change significantly during trains of one to thirty-one APs (P < 0.05). 4. Caffeine (10 mM), a known agonist of the ryanodine receptor, produced an increase in [Ca2+]i. The caffeine-induced rise in [Ca2+]i was attenuated (by > 90%) by lowering [Ca2+]o, and by ryanodine (10 microM), 2,5-di(t-butyl)hydroquinone (DBHQ, 10 microM), or thapsigargin (100 nM). 5. Neurones incubated with ryanodine, DBHQ or thapsigargin required at least eight APs to evoke a detectable calcium transient. These reagents did not significantly affect Ca2+ influx (P < 0.05). In the presence of these inhibitors, the calcium transient-AP relation exhibited slopes of 1.2, 1.1 and 1.9 nM AP-1 for ryanodine, DBHQ and thapsigargin, respectively. When compared with the slope of 9.6 nM AP-1 in non-treated neurones, it appears that Ca2+ influx produced by a single AP is amplified by ca 5- to 10-fold.

Topics: Action Potentials; Animals; Cadmium; Caffeine; Calcium; Calcium-Transporting ATPases; Central Nervous System Stimulants; Electrophysiology; Enzyme Inhibitors; Female; Hydroquinones; Male; Neurons, Afferent; Nodose Ganglion; Rabbits; Ryanodine; Thapsigargin

The influence of the new calcium antagonist mibefradil (CAS 116666-63-8, Ro 405967) on calcium influx into rabbit smooth muscle cells (VSMC) was studied. Angiotensin II-stimulated divalent cations entry was blocked by mibefradil at micromolar concentrations (1-10 mumol/l). In the same range of concentrations, the antagonist depressed capacitative calcium influx evoked by thapsigargin- and 2,5-di-tert-butylhydroquinone (BHQ)-dependent depletion of internal depots. The ability to block the receptor-mediated pathway of calcium current into VSMC may explain in part the difference between the mode of pharmacological action of mibefradil as compared to other calcium antagonists.

Topics: Angiotensin II; Animals; Benzimidazoles; Blood Platelets; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cell Membrane; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Hydroquinones; In Vitro Techniques; Membrane Potentials; Mibefradil; Muscle, Smooth, Vascular; Rabbits; Tetrahydronaphthalenes; Thapsigargin; Vasoconstrictor Agents

Central to the pathophysiology of stenosis following balloon angioplasty and arterial bypass surgery is proliferation of vascular smooth muscle cells (VSMCs). To investigate the role of calcium (Ca2+) in VSMC proliferation, the effect of thapsigargin, Ca2+ ionophore A23187, ionomycin, cyclopiazonic acid and di-tert-butylhydroquinone (all of which raise intracellular Ca2+ levels) on the proliferation of cultured human VSMCs was observed.. Cultured VSMCs from human saphenous vein were treated with calcium-modulating drugs and proliferation was assessed by determining [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation and cell number.. Over a 48-h exposure, thapsigargin inhibited VSMC replication (median 50 per cent maximal inhibitory concentration 2 nmol/l) whereas the other drugs were much less effective. Short-term exposure (5, 10, 30 and 60 min) to thapsigargin elicited a significant dose-dependent inhibition of VSMC replication whereas, again, the other drugs were without significant effect.. Thapsigargin-sensitive intracellular Ca2+ pools play a key role in controlling VSMC proliferation and specialized means of administering thapsigargin may constitute a possible approach to preventing stenosis.

Topics: Adult; Aged; Calcimycin; Calcium; Cell Division; Cells, Cultured; Enzyme Inhibitors; Female; Humans; Hydroquinones; Indoles; Ionomycin; Male; Middle Aged; Muscle, Smooth, Vascular; Saphenous Vein; Thapsigargin; Varicose Veins

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+ levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+ levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+ levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.

Topics: Animals; Animals, Newborn; Calcium; Calcium-Transporting ATPases; Cell Differentiation; Coated Pits, Cell-Membrane; Coculture Techniques; Enzyme Inhibitors; Hematopoietic Stem Cells; Hydroquinones; Indoles; Ionomycin; Ionophores; Mice; Mice, Inbred Strains; Osteoclasts; Skull; Thapsigargin

Force of contraction (Fc) of isolated human and rat atrial myocardium shows characteristic patterns of mechanical restitution when single test intervals are interposed in regular stimulation. With several pharmacological agents that modify the function of the sarcoplasmic reticulum we have investigated the role of the sarcoplasmic reticulum in mechanical restitution in these two species. Caffeine, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) were used to reduce Ca2+ uptake, ryanodine to open Ca2+ release channels, and forskolin to stimulate Ca2+ uptake. Under control conditions, Fc recovered rapidly with test intervals shorter than steady-state, and was potentiated with longer than steady-state intervals. In human atrial tissue the maximum potentiation factor was 1.26 +/- 0.03 after a mean test interval of 9.70 +/- 1.55 s (n = 43) as compared to 3.07 +/- 0.45 after 30 sec. in rat atria (n = 48). Caffeine (3 mM) did not significantly affect steady-state Fc but abolished post-rest potentiation in human and rat preparations. Forskolin (1 microM) enhanced and accentuated the mechanical restitution curve in particular for short test intervals. In the presence of thapsigargin (10 microM), steady-state Fc and mechanical restitution could not be distinguished from time-matched controls exposed to solvent only, indicating that this agent is ineffective in human and rat atrial tissue. In contrast, the putative Ca2+ uptake inhibitor BHQ (100 microM) strongly reduced steady-state Fc and decreased potentiation at all intervals in human muscle, but shifted the mechanical restitution curve in parallel to lower values in rat atria. Ryanodine (10 nM) induced post-rest decay in human and depressed both steady-state Fc and post-rest potentiation in rat atrial muscle. From these results it is concluded that human and rat atrial muscle differ in the Ca2+ handling by the sarcoplasmic reticulum during mechanical restitution.

Topics: Animals; Binding, Competitive; Caffeine; Calcium; Calcium Channels; Colforsin; Enzyme Inhibitors; Humans; Hydroquinones; Myocardial Contraction; Phosphodiesterase Inhibitors; Rats; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin

Interferon-gamma (IFN-gamma) +/- tumor necrosis factor-alpha (TNF-alpha) induces antiproliferation and intracellular Ca2+ store depletion in a human submandibular ductal cell line (HSG), which can be reversed on cytokine removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 (Cell Physiol. 39): C514-C521, 1996]. Here we have examined a possible mechanism for the IFN-gamma-induced intracellular Ca2+ store depletion. There was a time-dependent decrease in thapsigargin-dependent internal Ca2+ release after exposure of the cells to the cytokines. The intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] protein in lysates and membranes of cells treated with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone, showed a similar time-dependent decrease (examined using a SERCA2 antibody). Removal of the cytokines, which resulted in recovery of cell growth and refill of internal Ca2+ stores, also increased the level of SERCA protein. The decrease in SERCA is not a result of decreased cell proliferation, since thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone, or serum-free growth conditions induced antiproliferative effects on HSG cells without any corresponding decrease in SERCA. We suggest that the IFN-gamma-induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+ stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-gamma.

Topics: Calcium; Calcium-Transporting ATPases; Cell Division; Cell Line; Cell Membrane; Culture Media, Serum-Free; Cytosol; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Hydroquinones; Interferon-gamma; Kinetics; Recombinant Proteins; Submandibular Gland; Thapsigargin; Time Factors; Tumor Necrosis Factor-alpha

In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA)-type Ca(2+)-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca(2+)-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca(2+)-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca(2+)-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; HL-60 Cells; Humans; Hydroquinones; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Neutrophils; Phagocytosis; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Vanadates

Because the role of intracellular Ca2+ in the two-signal process for the induction of nitric oxide (NO) synthesis is controversial, this study was undertaken to examine the role of Ca2+ in the transcriptional regulation of inducible NO synthase (iNOS) in murine peritoneal macrophages. Treatment of the cells with thapsigargin (TG) or 2,5-di-(t-butyl)-1,4-benzodihydroquinone (tBuBHQ), which are the specific and potent Ca(2+)-ATPase inhibitors of endoplasmic reticulum (ER), showed modest effects on tumoricidal function, whereas TG or tBuBHQ in combination with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) showed marked effects on tumoricidal function of the cells. The tumoricidal effects of the activated macrophages were correlated with the amount of NO synthesis, and totally abrogated by the use of NOS inhibitor, NG-monomethyl-L-arginine (NGMMA). The increases in NO synthesis was reflected as increased amounts of iNOS mRNA by Northern blotting. To confirm that iNOS induction was due to the changes in the intracellular Ca2+ level, the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca2+ chelator, was used. Blocking the increase of cytosolic free Ca2+ significantly decreased the induction of NO synthesis. To demonstrate that intracellular Ca2+ acts as a 'priming' signal rather than a 'triggering' signal on the induction of NO synthesis by murine peritoneal macrophages, we designed several experiments. When the cells were treated with TG 6 hr after the treatment with IFN-gamma, there was no increase in NO synthesis. In addition, when the cells were treated with TG or LPS 6 hr after treatment with tBuBHQ, a synergistic increase on NO synthesis was shown only in the case of LPS. When phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, was added to the cells 6 hr after the treatment with TG, there was a marked co-operative induction of NO synthesis, even though PMA alone has no effect. Based on the results obtained in this study, we suggest that cytosolic Ca2+ might be enough for the expression of iNOS gene as a priming signal and PKC might be involved in the induction of NO synthesis as a triggering signal by post-transcriptional modification of iNOS mRNA or iNOS itself in the activated murine peritoneal macrophages.

Topics: Animals; Blotting, Northern; Calcium; Cell Culture Techniques; Egtazic Acid; Female; Hydroquinones; Leukemia, Experimental; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Nitric Oxide; Terpenes; Thapsigargin

The effects of 2,5-di-tert-butylhydroquinone (DBHQ) and thimerosal on phosphatidylserine synthesis by the base exchange reaction and on calcium mobilization in intact glioma C6 cells were compared with that of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. It has been found that all these agents inhibit phosphatidylserine synthesis by 70%, but their effectiveness are different. The data show that this inhibition is caused by Ca2+ depletion of the endoplasmic reticulum, indicating that phosphatidylserine synthesis requires high concentration of Ca2+ within this structure. On this basis and on literature data, a new model for the localization of the serine base exchange enzyme in the endoplasmic reticulum membrane is proposed.

Topics: Brain Neoplasms; Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Glioma; Hydroquinones; Ionomycin; Phosphatidylserines; Terpenes; Thapsigargin; Thimerosal; Tumor Cells, Cultured

The biochemical transductional events involved in NO synthesis are not fully understood. These studies, therefore, were undertaken to elucidate the role of intracellular calcium and protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Thapsigargin (TG), Ca(2+)-ATPase inhibitor of endoplasmic reticulum, had modest activity on NO synthesis by itself, whereas phorbol ester, PKC activator, alone had no effect. When TG was used in combination with phorbol ester, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of phorbol ester was shown in the first 6 h after TG treatment. In addition, the ability of TG with phorbol ester on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca(2+)-ATPase, 2,5-di-(t-butyl)-1, 4-benzohydroquinone, and Ca2+ ionophore, A23187. This increase of NO synthesis was reflected as increased amount of NO synthase (NOS) mRNA, as determined by Northern blotting. Intracellular Ca2+ transient by TG was not affected in the presence or absence of extracellular Ca2+, indicating that TG must be effective on cytosolic Ca2+ pool. In addition, chelation of intracellular Ca2+ by acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular Ca2+ chelating agent, blocked TG- or TG + PMA-induced NO production. PKC inhibitors such as staurosporine or polymyxin B reduced only the synergistic cooperative effect of TG with phorbol ester without affecting TG-induced NO production. In addition, when the cells were pretreated with phorbol ester before TG treatment, there was no synergy between TG and phorbol ester, indicating that PKC is not directly involved in the expression of NOS but involved in "triggering" signal. Secretion of NO corresponded with tumor cell killing, but TG plus phorbol ester-activated macrophages failed to kill tumor cell targets in the presence of Ng-monomethyl-L-arginine. Collectively, these data illustrate that mobilization of intracellular Ca2+ provides a "priming" signal for induction of NOS gene expression by itself and it also requires PKC as a "triggering" signal for macrophage tumoricidal activity.

Topics: Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Chelating Agents; Cytotoxicity, Immunologic; Drug Synergism; Egtazic Acid; Humans; Hydroquinones; Interferon-gamma; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Nitric Oxide; Protein Kinase C; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

An ER/SR Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), was found to suppress apoptotic cell death of IL-3-dependent cell lines, FDC.P2, IC-2, and Ba/F3, upon IL-3 deprivation. Structurally unrelated ER/SR Ca2+-ATPase inhibitors, thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone also maintained cell viability in the absence of IL-3. In the Ca2+-free medium CPA failed to suppress apoptosis, suggesting that the anti-apoptotic activity of CPA is dependent on extracellular calcium. The culture supernatant of CPA-treated cells was able to prolong cell survival of FDC.P2 in the absence of IL-3. An anti-IL-4 antibody almost completely eliminated the anti-apoptotic activity of CPA. Indeed, a significant amount of IL-4 was detected in the supernatant of cells treated with ER/SR Ca2+-ATPase inhibitors. Thus, our present data clearly demonstrate not only that ER/SR Ca2+-ATPase inhibitors induce the secretion of IL-4 in IL-3-dependent cell lines, but also that IL-4 can replace IL-3 in protecting these cells from apoptotic cell death in an autocrine manner.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Calcium-Transporting ATPases; Cell Line; Cell Survival; Culture Media, Conditioned; Endoplasmic Reticulum; Enzyme Inhibitors; Hydroquinones; Indoles; Interleukin-3; Interleukin-4; Mice; Sarcoplasmic Reticulum; Thapsigargin

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Chelating Agents; Cytosol; Dictyostelium; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix Proteins; Fungal Proteins; Gene Expression Regulation, Developmental; Hexanones; Hydroquinones; Protozoan Proteins; Recombinant Fusion Proteins; Thapsigargin; Transcriptional Activation

It has been suggested that excitation in Drosophila photoreceptors may be mediated by the depletion of intracellular Ca2+ stores (capacitative Ca2+ entry). To investigate this hypothesis, simultaneous whole-cell recordings and Indo-1 Ca2+ measurements were made from dissociated Drosophila photoreceptors, whilst testing the effects of Ca2+ releasing agents. In Ca2+ free Ringer's solution, thapsigargin raised cytosolic Ca2+ by approximately 80 nM; subsequent application of ionomycin released further Ca2+ (approximately 100 nM). A possible third compartment was indicated by the ability of monensin to mobilize further Ca2+ after saturating doses of ionomycin. Under most conditions, none of these agents activated an inward conductance, and their effects on the light response were consistent with their effects on cytosolic Ca2+. However, in the absence of both external Ca2+ and Mg2+ (to relieve a Mg2+ block of the light-sensitive channels), and after loading cells with BAPTA buffering cytosolic free Ca2+ at approximately 10 nM, ionomycin (but not thapsigargin) activated inward currents of approximately 800 pA. The response to ionomycin was enhanced (10 nA) by buffering cytosolic Ca2+ at 250 nM. A similar current also developed after approximately 3 min in cells loaded with Ca-BAPTA without any ionomycin application. The current-voltage relationships of currents activated by Ca-BAPTA or ionomycin were indistinguishable from that of the light-activated conductance and were similarly affected by a null mutation of the transient receptor potential (trp) gene which is believed to encode a subunit of the light-sensitive channels. These experiments provide some evidence for the suggestion that the light-activated and trp-dependent conductance in Drosophila photoreceptors can be activated by depletion of internal stores. However, activation by Ca-BAPTA and ionomycin had an absolute requirement for cytosolic Ca2+ as no currents could be activated by ionomycin in cells loaded with BAPTA and no Ca2+.

Topics: Animals; Calcium; Calcium Channels; Calcium-Transporting ATPases; Chelating Agents; Drosophila; Egtazic Acid; Electric Conductivity; Electrophysiology; Enzyme Inhibitors; Hydroquinones; Ionomycin; Ionophores; Isotonic Solutions; Magnesium; Monensin; Mutagenesis; Photoreceptor Cells, Invertebrate; Ringer's Solution; Signal Transduction; Thapsigargin; TRPC Cation Channels

1. Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (t-BuBHQ). 2. Cytosolic Ca2+ concentration was evaluated with Fura-2, in the absence of Ca2+ influx. Addition of 5 microM NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 microM) did not induce significant modifications. This NO-induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+]. 3. NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml-1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 microM thapsigargin or by 20 microM t-BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2-dependent self-amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO-insensitive Ca2+ stores. 4. These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO-sensitive internal stores evoked by stimulation of receptors or by Ca(2+)-ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.

Topics: Adult; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Humans; Hydroquinones; In Vitro Techniques; Male; Nitric Oxide; Stimulation, Chemical; Thapsigargin; Thrombin

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

Topics: Animals; Basophils; Calcium; Calcium-Transporting ATPases; Cell Degranulation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroquinones; Indoles; Leukemia, Basophilic, Acute; Leukotriene C4; Lipoxygenase Inhibitors; Mice; Naphthalenes; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Xanthine oxidase inhibits agonist-stimulated Ca2+ signaling in calf pulmonary artery endothelial cells by an H2O2-dependent mechanism. We investigated the effect of xanthine oxidase on luminal Ca2+ content of the inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ store.. Luminal Ca2+ content was estimated from the net release of Ca2+ activated by 2,5-di-t-butylhydroquinone (BHQ), an inhibitor of microsomal Ca2+ pumps.. Initially, xanthine oxidase depleted the IP3-sensitive Ca2+ store of releasable Ca2+, but with more prolonged incubation, the enzyme also depleted non-IP3-sensitive stores. In addition, xanthine oxidase inhibited capacitative Ca2+ influx. Similar results were observed when thapsigargin was substituted for BHQ.. Depletion of luminal Ca2+ content within the IP3-sensitive Ca2+ store contributes to xanthine oxidase inhibition of Ca2+ signaling in vascular endothelial cells.

Topics: Animals; Calcium; Cattle; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Hydrogen Peroxide; Hydroquinones; Inositol 1,4,5-Trisphosphate; Reactive Oxygen Species; Terpenes; Thapsigargin; Xanthine Oxidase

The ability of putative Ca(2+)-ATPase inhibitor of endoplasmic reticulum (ER), thapsigargin (TG), to induce nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. TG alone had small effect on NO synthesis, whereas TG in combination with LPS markedly increased NO synthesis in a dose dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA by Northern blotting. In addition, the ability of TG on NO synthesis could be mimicked by another chemically unrelated inhibitor of Ca(2+)-ATPase, 2,5-DI-(t-butyl)-1, 4-benzohydroquinone (tBuBHQ). Adding EGTA, a calcium chelator, to the incubation medium significantly reduced the ability of macrophages to induce NO synthesis in response to the optimal stimulation of TG or TG plus LPS. These results therefore demonstrate that intracellular Ca2+ pool depletion is linked to the induction of NO synthesis in murine peritoneal macrophages and further suggest that it is also related with interferon-gamma (IFN-gamma)-induced signaling.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Chelating Agents; Egtazic Acid; Endoplasmic Reticulum; Enzyme Induction; Enzyme Inhibitors; Female; Hydroquinones; Interferon-gamma; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C3H; Nitric Oxide; Nitric Oxide Synthase; RNA, Messenger; Terpenes; Thapsigargin

Thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, depletes intracellular Ca2+ stores and induces a sustained Ca2+ influx without altering phosphatidyl inositol levels. TG plus phorbol myristate acetate (PMA) but not TG alone induced IL-2 in Jurkat T cells, suggesting that TG had no effect on protein kinase C (PKC). However, TG induced increases in IL-2R alpha protein as well as IL-2R alpha mRNA in Jurkat T cells in a dose-dependent manner. A similar increase in IL-2R alpha by TG was also observed in human peripheral T cells. Further, like PMA, TG markedly induced NF kappa B in Jurkat T cells. However, TG and PMA exhibited a synergistic action on IL-2R alpha expression, suggesting that TG and PMA induce IL-2R alpha through distinct pathways. PMA- but not TG-induced IL-2R alpha is inhibited by the PKC inhibitor H7, whereas TG- but not PMA-induced IL-2R alpha was inhibited by cholera toxin, forskolin and 1,9-dideoxy forskolin. In toto, these results suggest that TG induces IL-2R alpha in human T cells through a PKC-independent pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Base Sequence; Calcimycin; Calcium; Calcium-Transporting ATPases; Cholera Toxin; Colforsin; Drug Synergism; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Hydroquinones; Isoquinolines; Leukemia-Lymphoma, Adult T-Cell; Molecular Sequence Data; Neoplasm Proteins; NF-kappa B; Piperazines; Protein Kinase C; Receptors, Interleukin-2; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Staurosporine; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Bone Neoplasms; Calcium; Calcium-Transporting ATPases; Cell Membrane; Cell Membrane Permeability; Cytosol; Egtazic Acid; Endoplasmic Reticulum; Fura-2; Hydroquinones; Inositol 1,4,5-Trisphosphate; Nifedipine; Osteoblasts; Osteosarcoma; Terpenes; Thapsigargin; Thrombin; Tumor Cells, Cultured

In the absence of extracellular Ca2+, extensive Ca2+ release from the platelet intracellular stores [monitored as an increase of intracellular Ca2+ concentration ([Ca2+]i)] is produced by the combined action of the endomembrane Ca(2+)-ATPase inhibitor thapsigargin and 2 nM ionomycin. The titration of Ca2+ unloading with thapsigargin (plus ionomycin) shows that a substantial fraction of the store-associated Ca2+ is released by 8-10 nM thapsigargin, but that 100-200 nM thapsigargin is required for the complete release. The store depletion obtained in similar conditions with a different endomembrane Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ), is always incomplete. It is completed by thrombin or by 10 nM thapsigargin. We conclude that two different types of Ca2+ pumps exist in platelets, one sensitive to TBHQ and to high thapsigargin, the other insensitive to TBHQ and sensitive to low thapsigargin. They are distributed separately in discrete subpopulations of the agonist-sensitive stores. The influx of external Ca2+ is maximal when both types of stores are Ca(2+)-depleted, either by high thapsigargin or by the combined action of low thapsigargin and TBHQ.

Topics: Antioxidants; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cytosol; Dose-Response Relationship, Drug; Humans; Hydroquinones; In Vitro Techniques; Ionomycin; Kinetics; Terpenes; Thapsigargin

We have studied the role of intracellular calcium sequestration on human immunodeficiency virus (HIV) production by latently infected T-lymphocytic cells. Inhibition of the sarco-endoplasmic reticulum-type calcium transport ATPases by thapsigargin or cyclopiazonic acid induced activation of HIV production in the CEM-derived ACH-2 cells. An approximately 50% depletion of the thapsigargin-sensitive calcium pools as measured fluorimetrically of Indo-loaded cells fully activated virus production. Viral activation was manifest by increases in soluble viral core p24 production, increases in cellular immunofluorescent staining for viral antigens, and increased viral transcription as measured by HIV long terminal repeat-directed expression of the chloramphenicol acetyltransferase reporter gene. Virus induction could be blocked in a dose-dependent manner by the calcium channel blocker econazole. Virus production by the Jurkat-derived HIV-1-inducible J1.1 cells was not significantly stimulated by thapsigargin. These data indicate that intracellular calcium pool function is involved in the control of the transcription of proviral HIV in a cell type-specific manner within the T-lymphoid lineage and that ACH-2 cells represent a useful model for the study of calcium dependent activation of the transcription of proviral HIV.

Topics: Calcium; Calcium-Transporting ATPases; Cell Line; Chloramphenicol O-Acetyltransferase; Fluorescent Antibody Technique; HIV-1; Humans; Hydroquinones; Indoles; Kinetics; Terpenes; Thapsigargin; Virus Replication

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Arachidonic Acid; Calcium; CHO Cells; Cricetinae; Drug Synergism; Enzyme Activation; Hydroquinones; Inositol 1,4,5-Trisphosphate; Peptide YY; Peptides; Pertussis Toxin; Phenylephrine; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Protein Kinase C; Receptors, Adrenergic, alpha-1; Receptors, Neuropeptide Y; Recombinant Fusion Proteins; Signal Transduction; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Virulence Factors, Bordetella

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Antioxidants; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Channel Agonists; Calcium-Transporting ATPases; Endothelins; Fatty Acids, Unsaturated; Fura-2; Humans; Hydrazines; Hydroquinones; In Vitro Techniques; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Endothelin; Receptors, Thromboxane; Terpenes; Thapsigargin; Thrombin; Thromboxane A2; Vasoconstrictor Agents

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Biological Transport; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Hydroquinones; Indoles; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Potassium; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

1. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in single cells isolated from adult rat supraoptic (SO) nuclei. The great majority of cells (85%) were neurones and most were immunoreactive to oxytocin or to vasopressin (AVP). 2. The resting [Ca2+]i of the majority (80%) of the neurones remained stable while 20% of the neurones displayed spontaneous [Ca2+]i oscillations which disappeared in low-Ca2+ (100 nM) EGTA buffer. 3. Addition of 100 nM oxytocin increased the [Ca2+]i in both stable and oscillating cells. Two types of responses were observed: (i) a sustained response with [Ca2+]i being maintained at an elevated level and (ii) a brief response with [Ca2+]i quickly returning to a near-resting level. Responses were reproducible, dose dependent and blocked with a specific oxytocin antagonist. 4. Removal of extracellular Ca2+ did not block the oxytocin response. In EGTA buffer, application of thapsigargin (200 nM) onto oxytocin-sensitive cells induced an increase in [Ca2+]i and inhibited the oxytocin response. These effects were not induced by other intracellular Ca2+ mobilizers such as tBuBHQ (see Methods) or caffeine. 5. In conclusion, half of the SO cells respond to oxytocin with a rise in [Ca2+]i. The effect is mediated by oxytocin receptors and results from release of Ca2+ from thapsigargin-sensitive stores.

Topics: Animals; Antioxidants; Arginine Vasopressin; Caffeine; Calcium; Calcium-Transporting ATPases; Female; Fura-2; Hydroquinones; Immunohistochemistry; In Vitro Techniques; Male; Oxytocin; Rats; Rats, Wistar; Staining and Labeling; Supraoptic Nucleus; Terpenes; Thapsigargin; Vasotocin

The specific inhibitors of the endoplasmic reticulum Ca2+ pump, thapsigargin and 2,5-di-tert-butylhydroquinone (DBHQ), stimulated reinitiation of DNA synthesis in synergy with either phorbol 12,13-dibutyrate or bombesin in Swiss 3T3 cells. Maximum stimulation was achieved at 0.5 nM thapsigargin and 7.5 microM DBHQ. Kinetics of [3H]thymidine incorporation were consistent with exit from G0 and entry into S phase. Autoradiography of labeled nuclei showed that the increase in [3H]thymidine incorporation was due to an increase in the proportion of cells entering into DNA synthesis. Down-regulation or selective inhibition of protein kinase C abolished this synergistic stimulation of DNA synthesis. Thapsigargin and DBHQ did not potentiate protein kinase C-mediated signals such as direct phosphorylation of myristoylated alanine-rich C-kinase substrate, activation of mitogen-activated protein kinase, and tyrosine phosphorylation of bands 110,000-130,000 and 70,000-80,000. Thapsigargin and DBHQ caused a marked reduction in the ability of bombesin to induce a rapid and transient increase in intracellular Ca2+ via depletion of total cellular Ca2+, measured by 45Ca2+ content. The synergistic stimulation of DNA synthesis by DBHQ and phorbol 12,13-dibutyrate was dependent on a high concentration of extracellular Ca2+ (ED50 = 410 microM) and was preferentially inhibited by the inhibitor of Ca2+ influx econozole. This suggests a role for Ca2+ entry in growth control. This is the first time that either thapsigargin or DBHQ has been shown to stimulate the reinitiation of DNA synthesis in any target cell.

Topics: 3T3 Cells; Animals; Antioxidants; Bombesin; Calcium; Calcium-Transporting ATPases; DNA Replication; Drug Synergism; Hydroquinones; Mice; Mitogens; Phorbol 12,13-Dibutyrate; Terpenes; Thapsigargin

The calcium-mobilizing agents thapsigargin and 2,5-di-(tert-butyl)-1,4- benzohydroquinone were shown to markedly elevate the intracellular calcium concentration of chick embryo chondrocytes in a dose-dependent manner. Under these conditions, the metabolism of macromolecules was variably affected. The synthesis and secretion of protein in general, and of collagen in particular, were significantly inhibited; in contrast, proteoglycan synthesis (but not glycosaminoglycan synthesis) was inhibited, whereas secretion was unaffected. Flunarizine, which prevented the thapsigargin-induced intracellular calcium elevation, and EGTA, which caused only a transient thapsigargin-induced intracellular calcium elevation, did not reverse these alterations. It was concluded, therefore, that the observed effects of thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone on chondrocyte macromolecule metabolism were not related to the ability of these drugs to increase the cytosolic free calcium concentration but may have been due to the specific depletion of the calcium sequestered in the endoplasmic reticulum. The differential effect of these drugs on protein and proteoglycan secretion suggests that the intracellular trafficking of these two classes of macromolecules may be controlled independently.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cartilage; Cells, Cultured; Chick Embryo; Collagen; Flunarizine; Hydroquinones; Proteins; Proteoglycans; Terpenes; Thapsigargin; Time Factors

Gonadotropin-releasing hormone induces oscillatory release of Ca2+ from inositol trisphosphate-sensitive stores of gonadotropes. Simultaneously with electrophysiological measures of cytoplasmic [Ca2+], corresponding changes in [Ca2+] within intracellular stores were monitored with a fluorescent dye, mag-indo-1. Each cycle of oscillation released only 10% of the detectable stored Ca2+. Some Ca2+ was recovered by the stores using a mechanism sensitive to inhibitors of intracellular Ca2+ ATPases, and much of the remainder was temporarily and rapidly pumped into other intracellular compartments or out of the cell. The dynamics of Ca2+ oscillations are thus more complex than a repeated emptying and refilling of a single compartment. The free concentrations measured show that intracellular Ca2+ store compartments contain strong Ca2+ buffers.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Gonadotropin-Releasing Hormone; Hydroquinones; In Vitro Techniques; Kinetics; Male; Oscillometry; Pituitary Gland, Anterior; Rats; Rats, Sprague-Dawley; Terpenes; Thapsigargin; Time Factors

The role of heterotrimeric GTP-binding proteins in the process of store-operated Ca2+ inflow in hepatocytes was investigated by testing the ability of pertussis toxin to inhibit thapsigargin- and 2,5-di-tert-butylhydroquinone (DBHQ)-induced bivalent cation inflow. Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited markedly inhibited rates of both Ca2+ and Mn2+ inflow when these were stimulated by vasopressin, angiotension II, epidermal growth factor, thapsigargin and DBHQ. Pertussis toxin had little effect on the basal intracellular free Ca2+ concentration ([Ca2+]i), basal rates of Ca2+ and Mn2+ inflow, the abilities of vasopressin, angiotensin II, thapsigargin and DBHQ to induce the release of Ca2+ from intracellular stores, and the maximum value of [Ca2+]i reached following agonist-induced release of Ca2+ from intracellular stores. It is concluded that store-operated Ca2+ inflow in hepatocytes employs a slowly ADP-ribosylated trimeric GTP-binding protein and is the physiological mechanism, or one of the physiological mechanisms, by which vasopressin and angiotensin stimulate plasma membrane Ca2+ inflow in this cell type.

Topics: Angiotensin II; Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Calibration; Epidermal Growth Factor; GTP-Binding Proteins; Hydroquinones; Injections, Intraperitoneal; Liver; Manganese; Pertussis Toxin; Rats; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Vasopressins; Virulence Factors, Bordetella

The mechanism of store-operated Ca2+ inflow in hepatocytes was investigated using fluo-3 and fura-2 to monitor changes in the concentration of intracellular free Ca2+ in single cells, and 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-diphosphate, P4(5)-1-(2-nitrophenyl)ethyl ester ('caged' GPIP2) and 'caged' guanosine 5'-[gamma thio]triphosphate (GTP gamma S) (introduced into the cytoplasmic space by microinjection), thapsigargin and 2,5-di-tert- butylhydroquinone (DBHQ) to stimulate Ca2+ inflow. Photolysis of 'caged' GPIP2 or 'caged' GTP gamma S stimulated Ca2+ inflow. The abilities of GPIP2, thapsigargin and DBHQ to stimulate Ca2+ inflow were inhibited by the pre-treatment of hepatocytes with pertussis toxin in vivo for 36 h. Thapsigargin-stimulated Ca2+ inflow was also inhibited by guanosine 5'-[beta-thio]diphosphate (GDP beta S) (introduced by microinjection). It is concluded that, in hepatocytes, store-operated Ca2+ inflow induced by the actions of either inositol 1,4,5-trisphosphate, thapsigargin or DBHQ requires a pertussis toxin-sensitive trimeric G-protein.

Topics: Aniline Compounds; Animals; Calcium; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Hydroquinones; Inositol Phosphates; Liver; Macromolecular Substances; Pertussis Toxin; Phosphatidylinositol Phosphates; Photolysis; Rats; Terpenes; Thapsigargin; Thionucleotides; Virulence Factors, Bordetella; Xanthenes

In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

Topics: Animals; Biological Transport; Calcium; Esters; Fura-2; Hydroquinones; Inositol 1,4,5-Trisphosphate; Liver; Male; Manganese; Microinjections; Rats; Rats, Wistar; Receptors, Cell Surface; Salts; Signal Transduction; Spectrometry, Fluorescence; Terpenes; Thapsigargin

Treatment of T lymphoblasts with stimuli that mobilize [Ca2+]i, such as ionophores (ionomycin and A23187) and endoplasmic reticulum Ca(2+)-ATPase inhibitors (thapsigargin, 2,5-di-(tert.-butyl)-hydroquinone and cyclopiazonic acid), activated T cell binding to extracellular matrix (ECM) proteins. T lymphoblast adhesion to ECM proteins stimulated by ionomycin, thapsigargin, or PMA was inhibited by an anti-beta 1 integrin mAb (4B4), confirming the role of beta 1 integrins in regulated T cell-ECM interactions. Study of the alpha integrin subunit specificity of activated lymphoblast-fibronectin interactions demonstrated that alpha 5 beta 1 was the major integrin receptor regulating binding to fibronectin. These results indicate that intracellular Ca2+ mobilization plays a major contributory role in the activation of T cell beta 1 integrins.

Topics: Antibodies, Monoclonal; Antioxidants; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Adhesion; Cells, Cultured; Collagen; Endoplasmic Reticulum; Fibronectins; Humans; Hydroquinones; Indoles; Integrin beta1; Integrins; Ionomycin; Laminin; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of vascular endothelial cells after selective inhibition of the endoplasmic-reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinone (BHQ) increases Ca2+ influx from the extracellular space in the absence of phosphoinositide hydrolysis. One model to account for these results suggests a close association between the internal store and the plasmalemma, allowing for the vectorial movement of Ca2+ from the extracellular space to the ER. Furthermore, recent evidence suggests that Ins(1,4,5)P3-induced Ca2+ release from intracellular stores is regulated by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-induced Ca2+ entry may directly regulate Ca2+ release from internal stores. To test these hypotheses, we examined the effect of 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF 96365), an inhibitor of Ca2+ influx, on unidirectional 45Ca2+ efflux (i.e. retrograde radioisotope flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting release of Ca2+ from internal stores, and a sustained increase indicative of Ca2+ influx. In the absence of agonist, 45Ca2+ efflux was slow and monoexponential with time. Addition of BK dramatically increased 45Ca2+ efflux; 50-60% of the 45Ca2+ associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+]i and the stimulation of 45Ca2+ efflux was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM), SKF 96365 (50 microM) inhibited the rise in [Ca2+]i attributed to influx without affecting release from internal stores. At a threshold concentration of bradykinin (2 nM), SKF 96365 blocked influx, but stimulated Ca2+ release from internal stores, as indicated by increases in both the transient component of the fura-2 response and 45Ca2+ efflux. Thapsigargin (200 nM) and BHQ (10 microM) produced an increase in 45Ca2+ efflux that was completely blocked by SKF 96365 or by cytosolic loading with BAPTA. These results suggest the existence of a restricted sub-plasmalemmal space that is defined by an area of surface membrane which contains the Ca(2+)-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca(2+)-pump units.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium-Transporting ATPases; Cattle; Cell Compartmentation; Cells, Cultured; Cytoplasm; Egtazic Acid; Endoplasmic Reticulum; Endothelium, Vascular; Extracellular Space; Humans; Hydroquinones; Imidazoles; Inositol 1,4,5-Trisphosphate; Terpenes; Thapsigargin

Autophagic sequestration of endogenous lactate dehydrogenase or electroinjected [3H]raffinose in isolated rat hepatocytes was strongly suppressed by the Ca2+ chelator EGTA, unless the cells had previously been electroloaded in the presence of high concentrations of Ca2+ (1.2 mM). The extracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and the intracellular Ca2+ chelator BAPTA/tetra(acetoxymethyl)-ester (BAPTA/AM) both inhibited autophagy to the same extent as did EGTA. Inhibitors of Ca(2+)-activated protein kinases (KN-62, H-7, W-7) had little or no effect on autophagy, indicating that the Ca2+ requirement of autophagy was not mediated by such kinases. Agents that elevate cytosolic Ca2+ by releasing Ca2+ from intracellular stores, like thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and the ionophores A23187 and ionomycin, inhibited autophagy strongly, implicating depletion of sequestered rather than of cytosolic intracellular Ca2+ as a common mechanism of inhibition. Lysosomal (propylamine-sensitive) protein degradation, known to be largely autophagy-dependent, was inhibited by thapsigargin and tBuBHQ. Thapsigargin had no effect on cellular ATP levels, but all agents tested (thapsigargin, tBuBHQ, ionophores) inhibited protein synthesis. Our results suggest that autophagy, like protein synthesis, is dependent on the presence of Ca2+ in some intracellular storage compartment.

Topics: Animals; Autophagy; Calcimycin; Calcium; Calcium-Transporting ATPases; Chelating Agents; Hydroquinones; In Vitro Techniques; Ionomycin; Liver; Male; Protein Kinase Inhibitors; Rats; Rats, Wistar; Terpenes; Thapsigargin

In Jurkat cells, the three Ca(2+)-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-tert-butylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca(2+)-ATPase inhibitors did not induce the formation of inositol trisphosphate from the hydrolysis of phosphatidylinositides. Emptying intracellular Ca2+ stores was accompanied by a decrease of phosphatidylserine (PtdSer) synthesis as previously observed in PHA- or CD3 mAb-treated Jurkat cells. In the presence of a phorbol ester able to activate protein kinase C, TPA, the three Ca(2+)-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca(2+)-ATPase inhibitors. In purified human peripheral blood T lymphocytes, the same inhibitors induced moderate if any cytosolic Ca2+ rise, in the absence of external calcium. Nevertheless analysis of PtdSer synthesis suggested that intracellular stores were efficiently depleted by DtBuHQ and cyclopiazonic acid but not by thapsigargin. In contrast, the three compounds induced similar Ca2+ influx. However, in the presence of TPA, cyclopiazonic acid and DtBuHQ induce highly purified T cells to proliferate while thapsigargin did not, suggesting that the status of internal Ca2+ store may have a decisive role in T cell activation.

Topics: Calcium; Calcium-Transporting ATPases; Cell Division; Cell Line; Humans; Hydroquinones; Indoles; Interleukin-2; Lymphocyte Activation; Phorbol Esters; Phosphatidylserines; T-Lymphocytes; Terpenes; Thapsigargin

We characterized and directly compared the Ca(2+)-releasing actions of two inhibitors of endoplasmic-reticulum (ER) Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca2+]) was monitored by Ca(2+)-selective mini-electrodes. After ATP-dependent Ca2+ uptake, thapsigargin and tBuBHQ released Ca2+ with and EC50 of approximately 37 nM and approximately 2 microM respectively. Both agents mobilized Ca2+ predominantly from the Ins(1,4,5)P3-sensitive Ca2+ pool, and in this respect thapsigargin was more specific than tBuBHQ. The total increase in [Ca2+] obtained with thapsigargin and Ins(1,4,5)P3 was, on the average, only 7% greater than that with Ins(1,4,5)P3 alone. In contrast, the total increase in [Ca2+] obtained with tBuBHQ and Ins(1,4,5)P3 was 33% greater than that obtained with only InsP3 (P < 0.05). Although Ca2+ was rapidly mobilized by thapsigargin and tBuBHQ, complete depletion of the Ins(1,4,5)P3-sensitive Ca2+ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P3 induced additional Ca2+ release. The additional Ins(1,4,5)P3-induced Ca2+ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A1, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca(2+)-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P3-sensitive Ca2+ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca2+ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca2+ from the uptake compartment, whereas Ca2+ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P3.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Cell Membrane Permeability; Endoplasmic Reticulum; Hydroquinones; Inositol 1,4,5-Trisphosphate; Insulin; Insulin Secretion; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

The effects of the Ca(2+)-ATPase inhibitors thapsigargin (Tg) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca(2+)-regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca(2+)-ATPase activities of platelet mixed membranes, without any effect on the basal Mg(2+)-ATPase activity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM). The effect of the two inhibitors on 45Ca2+ release from saponin-permeabilized platelets has also been characterized. 45Ca2+ uptake into non-mitochondrial intracellular stores occurs via an ATP-dependent mechanism, and if added at equilibrium the second messenger Ins(1,4,5)P3 releases 50% of the accumulated 45Ca2+. Maximally effective concentrations of Tg (1 microM) and tBuBHQ (50 microM) release 77% and 68% of the accumulated 45Ca2+. Addition of Ins(1,4,5)P3 together with either Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-sensitive Ca2+ pool was a subset of the pool that is sensitive to the Ca(2+)-ATPase inhibitors. Release of 45Ca2+ by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca2+ release, and Tg was found not to affect [32P]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool without affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Both Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cells already treated with Tg or tBuBHQ produced further elevation of Ca2+, indicating agonist utilization of a Ca(2+)-ATPase inhibitor-insensitive pool. In aggregation experiments Tg and tBuBHQ showed different functional effects. In indomethacin-treated cells Tg induces slow aggregation and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kinase C activator dioctanoylglycerol (DiC8). Tg also showed greater ability than tBuBHQ to release [3H]arachidonic acid (AA) from [3H]AA-labelled platelets. Additionally, in [32P]Pi-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light

Topics: Binding Sites; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Humans; Hydroquinones; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Platelet Activation; Saponins; Terpenes; Thapsigargin

Using three putative, selective inhibitors of the Ca+(+)-pump ATPase of sarcoplasmic reticulum (SR), cyclopiazonic acid, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, the mechanisms of relaxation of the arterial smooth muscle by cyclic GMP-generating vasodilators were studied in the ring preparations of the rabbit aorta. Nitroglycerin (NTG) and atrial natriuretic factor (ANF) were used as representative cyclic GMP-generating vasodilators. When the above three inhibitors of SR Ca+(+)-pump ATPase were present during the period of reloading of intracellular store sites with Ca++, the phasic contractions induced by phenylephrine or caffeine in the succeeding period in Ca+(+)-free media containing 2 mM EGTA were inhibited in a concentration-dependent manner. With 3 x 10(-5) M of cyclopiazonic acid the inhibition was almost complete for both agonists. NTG and ANF relaxed the aorta contracted by phenylephrine (10(-6) M) and produced an increase in cyclic GMP content. All the three SR Ca+(+)-pump ATPase inhibitors produced a concentration-dependent inhibition of the relaxation by NTG and ANF without affecting the increment of cyclic GMP content. These results indicate that the proper functioning of SR Ca+(+)-pump ATPase is necessary for elicitation of relaxation by NTG and ANF. Enhanced sequestration of Ca++ by SR may be an important mechanism by which these compounds induce relaxation in this type of smooth muscle.

Topics: Adenosine Triphosphatases; Animals; Aorta; Atrial Natriuretic Factor; Calcium; Calcium-Transporting ATPases; Diltiazem; Drug Interactions; Hydroquinones; In Vitro Techniques; Indoles; Male; Muscle Contraction; Muscle Relaxation; Nitroglycerin; Phenylephrine; Rabbits; Sarcoplasmic Reticulum; Terpenes; Thapsigargin

Oxidant stress induced by t-butyl hydroperoxide (t-BuOOH) inhibits bradykinin-stimulated Ca2+ signalling in vascular endothelial cells. The effect of t-BuOOH on intracellular Ca2+ pools was determined by addition of Ca(2+)-releasing agents to fura-2-loaded cells suspended in Ca(2+)-free/EGTA buffer. In control cells, sequential additions of bradykinin and ionomycin produced similar increases in cytosolic free [Ca2+] ([Ca2+]i). By contrast, incubation with t-BuOOH progressively decreased the response of [Ca2+]i to bradykinin and increased that to ionomycin, suggesting that the total (ionomycin-releasable) Ca2+ pool remains replete during oxidant stress. The effect of t-BuOOH on the InsP3-sensitive Ca2+ pool was measured by the increase in [Ca2+]i or efflux of 45Ca2+ stimulated by 2,5-di-t-butylhydroquinone (BHQ). Incubation with t-BuOOH did not inhibit BHQ-stimulated increases in [Ca2+]i or 45Ca2+ efflux, suggesting that the InsP3-sensitive Ca2+ pool remains replete and releasable. Activity of the Ca(2+)-influx pathway stimulated by release of internal Ca2+ stores was determined via re-addition of Ca2+ to BHQ-stimulated cells suspended in Ca(2+)-free/EGTA buffer and via BHQ-stimulated 45Ca2+ uptake. Incubation of cells with t-BuOOH for 1 h significantly inhibited the influx pathway. At later time points, t-BuOOH increased basal [Ca2+]i and potentiated the response of [Ca2+]i to BHQ. Similar results were demonstrated with thapsigargin. Together, these findings suggest that (1) the inhibitory effect of t-BuOOH on bradykinin-stimulated release of Ca2+ from internal stores is not related to depletion of these stores, and (2) inhibition of the store-dependent Ca(2+)-influx pathway occurs by a direct effect of the influx pathway or by inhibition of the mechanism which links the internal Ca2+ store to plasmalemmal Ca2+ influx.

Topics: Animals; Antioxidants; Biological Transport; Bradykinin; Calcium; Cattle; Cells, Cultured; Endothelium, Vascular; Hydroquinones; Ionomycin; Oxidants; Peroxides; Terpenes; tert-Butylhydroperoxide; Thapsigargin

The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.

Topics: Animals; Blotting, Western; Calcium; Calcium-Transporting ATPases; Cations, Divalent; Cell Nucleus; Cross Reactions; Endoplasmic Reticulum; Hydrolysis; Hydroquinones; Intracellular Membranes; Rats; Terpenes; Thapsigargin

Receptor-mediated Ca2+ entry was studied in fura-2-loaded isolated hepatocytes. Emptying of internal Ca2+ stores by treatment with either the Ca(2+)-mobilizing hormone vasopressin or the inhibitors of the microsomal Ca2+ pump, 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) or thapsigargin, stimulated Ca2+ entry, as indicated by a rise in the cytosolic free Ca2+ concentration after Ca2+ was added to cells suspended in nominally Ca(2+)-free medium. The enhancement of Ca2+ entry was proportional to the degree of depletion of the intracellular Ca2+ pool and occurred also after removal of vasopressin from its receptor. In contrast, the stimulation of Mn2+ entry by vasopressin required the continuous presence of the agonist, since it was prevented by the addition of vasopressin receptor antagonist. This effect was observed under conditions where refilling of the agonist-sensitive pool was prevented by using nominally Ca(2+)-free medium. Unlike vasopressin, tBuBHQ or thapsigargin did not stimulate Mn2+ entry. These results suggest the existence of two pathways for receptor-mediated Ca2+ entry in hepatocytes, a 'capacitative' pathway that is sensitive to the Ca2+ content in the Ins(1,4,5)P3-sensitive Ca2+ pool and does not allow Mn2+ entry, and a second pathway that depends on receptor occupation, seems to require a second messenger for activation, and permits influx of Mn2+.

Topics: Animals; Calcium; Calcium Channels; Cell Membrane; Fura-2; Hydroquinones; Intracellular Fluid; Liver; Male; Manganese; Rats; Rats, Inbred Strains; Terpenes; Thapsigargin; Vasopressins

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.

Topics: Adenosine Diphosphate; Blood Platelets; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Compartmentation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Econazole; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Ion Channel Gating; Manganese; Miconazole; Osmolar Concentration; Platelet Activating Factor; Platelet Activation; Receptors, Cholinergic; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Terpenes; Thapsigargin; Thrombin; Vasopressins

Topics: Adenosine Triphosphate; Animals; Antioxidants; Calcium-Transporting ATPases; Hydroquinones; Kinetics; Muscles; Phosphorylation; Rabbits; Sarcoplasmic Reticulum; Terpenes; Thapsigargin

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.

Topics: Animals; Antioxidants; beta-Galactosidase; Calcimycin; Calcium; CHO Cells; Cholesterol; Cricetinae; Endoplasmic Reticulum; Homeostasis; Hydroquinones; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Ionomycin; Kinetics; Mevalonic Acid; Models, Biological; Recombinant Fusion Proteins; Signal Transduction; Sterols; Terpenes; Thapsigargin; Time Factors; Transfection

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

Topics: Antibodies, Monoclonal; Calcimycin; Calcium; CD3 Complex; Cell Membrane Permeability; Cells, Cultured; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Ionomycin; Lymphocyte Activation; Phosphatidylserines; T-Lymphocytes; Terpenes; Thapsigargin

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.

Topics: Biological Transport; Calcium; Calcium-Transporting ATPases; Cations, Divalent; Cell Line; Cell Membrane Permeability; Fura-2; Hydroquinones; Imidazoles; Indoles; Mycotoxins; N-Formylmethionine Leucyl-Phenylalanine; Terpenes; Thapsigargin

2,5-Di(tert-butyl)-1,4-benzohydroquinone has been shown to inhibit the Ca2+,M(2+)-ATPase of sarcoplasmic reticulum with an affinity of 0.4 microM. It has been shown to shift the E2-E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2-E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady-state level of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.

Topics: Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Hydroquinones; Kinetics; Sarcoplasmic Reticulum; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Vanadates

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca2+ mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the G alpha i2, G alpha i3, G alpha s, G beta 35, and G beta 36 protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca2+ mobilization, we investigated the effect of PT on Ca2+ responses using fluorescence spectroscopy and the Ca2+ indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 microgram/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca2+ from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca2+ release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca2+ response was inhibited by approximately 72%, suggesting that the major fraction of the Ca2+ response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the beta-subunit of G proteins because quantitative immunoblots showed that levels of the beta-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca2+ pool because Ca2+ release induced by agents that release Ca2+ directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [3H]inositol trisphosphate (IP3) formation because the level of inhibition of IP3 formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT-sensitive species is the major G protein pathway that couples thrombin-receptor activation to Ca2+ mobilization. This G protein appears to be involved not in the mechanisms that generate IP3 but rather possibly in coupling at the level of the intracellular Ca2+ store.

Topics: Adenosine Diphosphate Ribose; Animals; Antioxidants; Autoradiography; Blotting, Western; Calcium; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Hydroquinones; Inositol 1,4,5-Trisphosphate; Lanthanum; Muscle, Smooth, Vascular; Pertussis Toxin; Rats; Terpenes; Thapsigargin; Thrombin; Virulence Factors, Bordetella

Inhibitors of the endoplasmic reticulum Ca(2+)-ATPase like thapsigargin (TG) and 2,5-di (tert-butyl)-1,4-benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 microM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 microM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (Ki approximately 7 microM). Therefore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca(2+)-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca(2+)-ATPase inhibitors is a subset of the receptor sensitive calcium pool.

Topics: Arachidonic Acids; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cytosol; Endoplasmic Reticulum; Humans; Hydroquinones; Platelet Aggregation; Receptors, Cell Surface; Terpenes; Thapsigargin; Thrombin

The effects of two inhibitors of the microsomal Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone, were compared in hepatocytes and in a T-cell line (JURKAT). Both compounds mobilized the same intracellular Ca2+ pool, which contained the Ins(1,4,5)P3-sensitive store, in hepatocytes and in JURKAT cells. The mobilization of the internal Ca2+ store with either compound activated Mn2+ entry in JURKAT cells, but not in hepatocytes. This suggests different properties of the bivalent-cation entry pathway between these cell types.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Cell Line; Cells, Cultured; Cytosol; Fura-2; Humans; Hydroquinones; Kinetics; Leukemia, T-Cell; Liver; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Signal Transduction; Terpenes; Thapsigargin; Vasopressins

Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.

Topics: Antioxidants; Blood Platelets; Calcium; Calcium-Transporting ATPases; Fibrinolytic Agents; Humans; Hydroquinones; Nigericin; Platelet Activation; Prostaglandin-Endoperoxide Synthases; Sulfonamides; Terpenes; Thapsigargin; Thrombin; Thromboxane A2; Thromboxane B2

The regulation of Ca2+ uptake by receptors is incompletely understood. It has been proposed that the Ca2+ permeability of the plasma membrane increases in response to depletion of a critical intracellular Ca2+ storage compartment (Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W. (1989) J. Biol. Chem. 264, 12266-12271). This hypothesis is based largely on the effect of thapsigargin, an inhibitor of endomembrane CA(2+)-ATPases. Due to the existence of an endogenous leak, inhibition of Ca2+ uptake by thapsigargin induces depletion of the stores. This is accompanied by increased plasmalemmal Ca2+ permeability, without change in the level of inositol phosphates. On the other hand, depletion of the intracellular stores by 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ), a chemically unrelated inhibitor of the Ca(2+)-ATPases, fails to induce Ca2+ influx (Kass, G. E., Duddy, S. K., Moore, G. A., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). In an attempt to reconcile these observations, we analyzed in lymphocytes the mode of action of thapsigargin and BHQ. In addition, we tested the effects of cyclopiazonic acid (CPA), a blocker of the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase. All three compounds released Ca2+ from a common intracellular compartment. Thapsigargin and low concentrations of BHQ and CPA concomitantly elevated the plasmalemmal Ca2+ permeability. Higher concentrations of BHQ and CPA produced a secondary inhibition of the Ca2+ entry pathway, by a mechanism seemingly unrelated to their effects on the internal stores. This inhibitory side effect can account for the reported discrepancies between the effects of thapsigargin and BHQ. The data provide further support for the notion that endomembrane Ca2+ stores are functionally coupled to the plasma membrane Ca2+ permeability pathway.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Cell Membrane; Cell Membrane Permeability; Cytosol; Endoplasmic Reticulum; Hydroquinones; Indoles; Intracellular Membranes; Lymphocytes; Male; Manganese; Rats; Rats, Inbred Strains; Terpenes; Thapsigargin; Thymus Gland