Thapsigargin: A sesquiterpene lactone found in roots of THAPSIA. It inhibits CA(2+)-TRANSPORTING ATPASE mediated uptake of CALCIUM into SARCOPLASMIC RETICULUM.Terpenes: A class of compounds composed of repeating 5-carbon units of HEMITERPENES.Calcium-Transporting ATPases: Cation-transporting proteins that utilize the energy of ATP hydrolysis for the transport of CALCIUM. They differ from CALCIUM CHANNELS which allow calcium to pass through a membrane without the use of energy.Calcium: A basic element found in nearly all organized tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.Ionomycin: A divalent calcium ionophore that is widely used as a tool to investigate the role of intracellular calcium in cellular processes.Enzyme Inhibitors: Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.Endoplasmic Reticulum: A system of cisternae in the CYTOPLASM of many cells. In places the endoplasmic reticulum is continuous with the plasma membrane (CELL MEMBRANE) or outer membrane of the nuclear envelope. If the outer surfaces of the endoplasmic reticulum membranes are coated with ribosomes, the endoplasmic reticulum is said to be rough-surfaced (ENDOPLASMIC RETICULUM, ROUGH); otherwise it is said to be smooth-surfaced (ENDOPLASMIC RETICULUM, SMOOTH). (King & Stansfield, A Dictionary of Genetics, 4th ed)HydroquinonesInositol 1,4,5-Trisphosphate: Intracellular messenger formed by the action of phospholipase C on phosphatidylinositol 4,5-bisphosphate, which is one of the phospholipids that make up the cell membrane. Inositol 1,4,5-trisphosphate is released into the cytoplasm where it releases calcium ions from internal stores within the cell's endoplasmic reticulum. These calcium ions stimulate the activity of B kinase or calmodulin.Calcium Signaling: Signal transduction mechanisms whereby calcium mobilization (from outside the cell or from intracellular storage pools) to the cytoplasm is triggered by external stimuli. Calcium signals are often seen to propagate as waves, oscillations, spikes, sparks, or puffs. The calcium acts as an intracellular messenger by activating calcium-responsive proteins.Sarcoplasmic Reticulum Calcium-Transporting ATPases: Calcium-transporting ATPases that catalyze the active transport of CALCIUM into the SARCOPLASMIC RETICULUM vesicles from the CYTOPLASM. They are primarily found in MUSCLE CELLS and play a role in the relaxation of MUSCLES.Calcium Channels: Voltage-dependent cell membrane glycoproteins selectively permeable to calcium ions. They are categorized as L-, T-, N-, P-, Q-, and R-types based on the activation and inactivation kinetics, ion specificity, and sensitivity to drugs and toxins. The L- and T-types are present throughout the cardiovascular and central nervous systems and the N-, P-, Q-, & R-types are located in neuronal tissue.Fura-2: A fluorescent calcium chelating agent which is used to study intracellular calcium in tissues.Egtazic Acid: A chelating agent relatively more specific for calcium and less toxic than EDETIC ACID.Calcium Channel Blockers: A class of drugs that act by selective inhibition of calcium influx through cellular membranes.Estrenes: Unsaturated derivatives of the ESTRANES with methyl groups at carbon-13, with no carbon at carbon-10, and with no more than one carbon at carbon-17. They must contain one or more double bonds.Sarcoplasmic Reticulum: A network of tubules and sacs in the cytoplasm of SKELETAL MUSCLE FIBERS that assist with muscle contraction and relaxation by releasing and storing calcium ions.Ionophores: Chemical agents that increase the permeability of biological or artificial lipid membranes to specific ions. Most ionophores are relatively small organic molecules that act as mobile carriers within membranes or coalesce to form ion permeable channels across membranes. Many are antibiotics, and many act as uncoupling agents by short-circuiting the proton gradient across mitochondrial membranes.Caffeine: A methylxanthine naturally occurring in some beverages and also used as a pharmacological agent. Caffeine's most notable pharmacological effect is as a central nervous system stimulant, increasing alertness and producing agitation. It also relaxes SMOOTH MUSCLE, stimulates CARDIAC MUSCLE, stimulates DIURESIS, and appears to be useful in the treatment of some types of headache. Several cellular actions of caffeine have been observed, but it is not entirely clear how each contributes to its pharmacological profile. Among the most important are inhibition of cyclic nucleotide PHOSPHODIESTERASES, antagonism of ADENOSINE RECEPTORS, and modulation of intracellular calcium handling.Ryanodine: A methylpyrrole-carboxylate from RYANIA that disrupts the RYANODINE RECEPTOR CALCIUM RELEASE CHANNEL to modify CALCIUM release from SARCOPLASMIC RETICULUM resulting in alteration of MUSCLE CONTRACTION. It was previously used in INSECTICIDES. It is used experimentally in conjunction with THAPSIGARGIN and other inhibitors of CALCIUM ATPASE uptake of calcium into SARCOPLASMIC RETICULUM.Chelating Agents: Chemicals that bind to and remove ions from solutions. Many chelating agents function through the formation of COORDINATION COMPLEXES with METALS.Pyrrolidinones: A group of compounds that are derivatives of oxo-pyrrolidines. A member of this group is 2-oxo pyrrolidine, which is an intermediate in the manufacture of polyvinylpyrrolidone. (From Merck Index, 11th ed)Indoles: Benzopyrroles with the nitrogen at the number one carbon adjacent to the benzyl portion, in contrast to ISOINDOLES which have the nitrogen away from the six-membered ring.Boron Compounds: Inorganic or organic compounds that contain boron as an integral part of the molecule.Lanthanum: Lanthanum. The prototypical element in the rare earth family of metals. It has the atomic symbol La, atomic number 57, and atomic weight 138.91. Lanthanide ion is used in experimental biology as a calcium antagonist; lanthanum oxide improves the optical properties of glass.Cells, Cultured: Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.Calcimycin: An ionophorous, polyether antibiotic from Streptomyces chartreusensis. It binds and transports CALCIUM and other divalent cations across membranes and uncouples oxidative phosphorylation while inhibiting ATPase of rat liver mitochondria. The substance is used mostly as a biochemical tool to study the role of divalent cations in various biological systems.Inositol 1,4,5-Trisphosphate Receptors: Intracellular receptors that bind to INOSITOL 1,4,5-TRISPHOSPHATE and play an important role in its intracellular signaling. Inositol 1,4,5-trisphosphate receptors are calcium channels that release CALCIUM in response to increased levels of inositol 1,4,5-trisphosphate in the CYTOPLASM.Intracellular Fluid: The fluid inside CELLS.Cytosol: Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components.Thapsia: A plant genus of the family APIACEAE. Members contain THAPSIGARGIN and other guaianolides (SESQUITERPENES, GUAIANOLIDE).Macrocyclic Compounds: Cyclic compounds with a ring size of approximately 1-4 dozen atoms.Tunicamycin: An N-acetylglycosamine containing antiviral antibiotic obtained from Streptomyces lysosuperificus. It is also active against some bacteria and fungi, because it inhibits the glucosylation of proteins. Tunicamycin is used as tool in the study of microbial biosynthetic mechanisms.Carbachol: A slowly hydrolyzed CHOLINERGIC AGONIST that acts at both MUSCARINIC RECEPTORS and NICOTINIC RECEPTORS.TRPC Cation Channels: A subgroup of TRP cation channels that contain 3-4 ANKYRIN REPEAT DOMAINS and a conserved C-terminal domain. Members are highly expressed in the CENTRAL NERVOUS SYSTEM. Selectivity for calcium over sodium ranges from 0.5 to 10.Type C Phospholipases: A subclass of phospholipases that hydrolyze the phosphoester bond found in the third position of GLYCEROPHOSPHOLIPIDS. Although the singular term phospholipase C specifically refers to an enzyme that catalyzes the hydrolysis of PHOSPHATIDYLCHOLINE (EC, it is commonly used in the literature to refer to broad variety of enzymes that specifically catalyze the hydrolysis of PHOSPHATIDYLINOSITOLS.Calcium Radioisotopes: Unstable isotopes of calcium that decay or disintegrate emitting radiation. Ca atoms with atomic weights 39, 41, 45, 47, 49, and 50 are radioactive calcium isotopes.Ion Transport: The movement of ions across energy-transducing cell membranes. Transport can be active, passive or facilitated. Ions may travel by themselves (uniport), or as a group of two or more ions in the same (symport) or opposite (antiport) directions.Adenosine Triphosphate: An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.Manganese: A trace element with atomic symbol Mn, atomic number 25, and atomic weight 54.94. It is concentrated in cell mitochondria, mostly in the pituitary gland, liver, pancreas, kidney, and bone, influences the synthesis of mucopolysaccharides, stimulates hepatic synthesis of cholesterol and fatty acids, and is a cofactor in many enzymes, including arginase and alkaline phosphatase in the liver. (From AMA Drug Evaluations Annual 1992, p2035)Cell Line: Established cell cultures that have the potential to propagate indefinitely.Membrane Potentials: The voltage differences across a membrane. For cellular membranes they are computed by subtracting the voltage measured outside the membrane from the voltage measured inside the membrane. They result from differences of inside versus outside concentration of potassium, sodium, chloride, and other ions across cells' or ORGANELLES membranes. For excitable cells, the resting membrane potentials range between -30 and -100 millivolts. Physical, chemical, or electrical stimuli can make a membrane potential more negative (hyperpolarization), or less negative (depolarization).Fluorescent Dyes: Agents that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags.Kinetics: The rate dynamics in chemical or physical systems.Protein Kinase C: An serine-threonine protein kinase that requires the presence of physiological concentrations of CALCIUM and membrane PHOSPHOLIPIDS. The additional presence of DIACYLGLYCEROLS markedly increases its sensitivity to both calcium and phospholipids. The sensitivity of the enzyme can also be increased by PHORBOL ESTERS and it is believed that protein kinase C is the receptor protein of tumor-promoting phorbol esters.Endoplasmic Reticulum Stress: Various physiological or molecular disturbances that impair ENDOPLASMIC RETICULUM function. It triggers many responses, including UNFOLDED PROTEIN RESPONSE, which may lead to APOPTOSIS; and AUTOPHAGY.Dose-Response Relationship, Drug: The relationship between the dose of an administered drug and the response of the organism to the drug.Signal Transduction: The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway.Patch-Clamp Techniques: An electrophysiologic technique for studying cells, cell membranes, and occasionally isolated organelles. All patch-clamp methods rely on a very high-resistance seal between a micropipette and a membrane; the seal is usually attained by gentle suction. The four most common variants include on-cell patch, inside-out patch, outside-out patch, and whole-cell clamp. Patch-clamp methods are commonly used to voltage clamp, that is control the voltage across the membrane and measure current flow, but current-clamp methods, in which the current is controlled and the voltage is measured, are also used.Cell Membrane Permeability: A quality of cell membranes which permits the passage of solvents and solutes into and out of cells.Vanadates: Oxyvanadium ions in various states of oxidation. They act primarily as ion transport inhibitors due to their inhibition of Na(+)-, K(+)-, and Ca(+)-ATPase transport systems. They also have insulin-like action, positive inotropic action on cardiac ventricular muscle, and other metabolic effects.Bradykinin: A nonapeptide messenger that is enzymatically produced from KALLIDIN in the blood where it is a potent but short-lived agent of arteriolar dilation and increased capillary permeability. Bradykinin is also released from MAST CELLS during asthma attacks, from gut walls as a gastrointestinal vasodilator, from damaged tissues as a pain signal, and may be a neurotransmitter.Oxazoles: Five-membered heterocyclic ring structures containing an oxygen in the 1-position and a nitrogen in the 3-position, in distinction from ISOXAZOLES where they are at the 1,2 positions.Nifedipine: A potent vasodilator agent with calcium antagonistic action. It is a useful anti-anginal agent that also lowers blood pressure.Rabbits: The species Oryctolagus cuniculus, in the family Leporidae, order LAGOMORPHA. Rabbits are born in burrows, furless, and with eyes and ears closed. In contrast with HARES, rabbits have 22 chromosome pairs.Inositol Phosphates: Phosphoric acid esters of inositol. They include mono- and polyphosphoric acid esters, with the exception of inositol hexaphosphate which is PHYTIC ACID.Sodium-Calcium Exchanger: An electrogenic ion exchange protein that maintains a steady level of calcium by removing an amount of calcium equal to that which enters the cells. It is widely distributed in most excitable membranes, including the brain and heart.Biological Transport: The movement of materials (including biochemical substances and drugs) through a biological system at the cellular level. The transport can be across cell membranes and epithelial layers. It also can occur within intracellular compartments and extracellular compartments.Purinergic Agonists: Compounds that bind to and activate PURINERGIC RECEPTORS.Cell Membrane: The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.Caspase 12: A long pro-domain caspase that contains a caspase recruitment domain in its pro-domain region. Caspase 12 is activated by pro-apoptotic factors that are released during cell stress and by CARD SIGNALING ADAPTOR PROTEINS. It activates APOPTOSIS by cleaving and activating EFFECTOR CASPASES.Rats, Sprague-Dawley: A strain of albino rat used widely for experimental purposes because of its calmness and ease of handling. It was developed by the Sprague-Dawley Animal Company.Ryanodine Receptor Calcium Release Channel: A tetrameric calcium release channel in the SARCOPLASMIC RETICULUM membrane of SMOOTH MUSCLE CELLS, acting oppositely to SARCOPLASMIC RETICULUM CALCIUM-TRANSPORTING ATPASES. It is important in skeletal and cardiac excitation-contraction coupling and studied by using RYANODINE. Abnormalities are implicated in CARDIAC ARRHYTHMIAS and MUSCULAR DISEASES.Intracellular Membranes: Thin structures that encapsulate subcellular structures or ORGANELLES in EUKARYOTIC CELLS. They include a variety of membranes associated with the CELL NUCLEUS; the MITOCHONDRIA; the GOLGI APPARATUS; the ENDOPLASMIC RETICULUM; LYSOSOMES; PLASTIDS; and VACUOLES.Xanthenes: Compounds with three aromatic rings in linear arrangement with an OXYGEN in the center ring.Calcium Channel Agonists: Agents that increase calcium influx into calcium channels of excitable tissues. This causes vasoconstriction in VASCULAR SMOOTH MUSCLE and/or CARDIAC MUSCLE cells as well as stimulation of insulin release from pancreatic islets. Therefore, tissue-selective calcium agonists have the potential to combat cardiac failure and endocrinological disorders. They have been used primarily in experimental studies in cell and tissue culture.Enzyme Activation: Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.Tetradecanoylphorbol Acetate: A phorbol ester found in CROTON OIL with very effective tumor promoting activity. It stimulates the synthesis of both DNA and RNA.Staurosporine: An indolocarbazole that is a potent PROTEIN KINASE C inhibitor which enhances cAMP-mediated responses in human neuroblastoma cells. (Biochem Biophys Res Commun 1995;214(3):1114-20)Transcription Factor CHOP: A CCAAT-enhancer binding protein that is induced by DNA DAMAGE and growth arrest. It serves as a dominant negative inhibitor of other CCAAT-enhancer binding proteins.Tumor Cells, Cultured: Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely.Homeostasis: The processes whereby the internal environment of an organism tends to remain balanced and stable.Uridine Triphosphate: Uridine 5'-(tetrahydrogen triphosphate). A uracil nucleotide containing three phosphate groups esterified to the sugar moiety.Alkaloids: Organic nitrogenous bases. Many alkaloids of medical importance occur in the animal and vegetable kingdoms, and some have been synthesized. (Grant & Hackh's Chemical Dictionary, 5th ed)Gadolinium: Gadolinium. An element of the rare earth family of metals. It has the atomic symbol Gd, atomic number 64, and atomic weight 157.25. Its oxide is used in the control rods of some nuclear reactors.Imidazoles: Compounds containing 1,3-diazole, a five membered aromatic ring containing two nitrogen atoms separated by one of the carbons. Chemically reduced ones include IMIDAZOLINES and IMIDAZOLIDINES. Distinguish from 1,2-diazole (PYRAZOLES).Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone: A proton ionophore that is commonly used as an uncoupling agent in biochemical studies.Calcium Channels, L-Type: Long-lasting voltage-gated CALCIUM CHANNELS found in both excitable and nonexcitable tissue. They are responsible for normal myocardial and vascular smooth muscle contractility. Five subunits (alpha-1, alpha-2, beta, gamma, and delta) make up the L-type channel. The alpha-1 subunit is the binding site for calcium-based antagonists. Dihydropyridine-based calcium antagonists are used as markers for these binding sites.Econazole: An imidazole derivative that is commonly used as a topical antifungal agent.Aniline CompoundsRats, Wistar: A strain of albino rat developed at the Wistar Institute that has spread widely at other institutions. This has markedly diluted the original strain.Extracellular Space: Interstitial space between cells, occupied by INTERSTITIAL FLUID as well as amorphous and fibrous substances. For organisms with a CELL WALL, the extracellular space includes everything outside of the CELL MEMBRANE including the PERIPLASM and the cell wall.Barium: An element of the alkaline earth group of metals. It has an atomic symbol Ba, atomic number 56, and atomic weight 138. All of its acid-soluble salts are poisonous.Ruthenium Red: An inorganic dye used in microscopy for differential staining and as a diagnostic reagent. In research this compound is used to study changes in cytoplasmic concentrations of calcium. Ruthenium red inhibits calcium transport through membrane channels.Cell Compartmentation: A partitioning within cells due to the selectively permeable membranes which enclose each of the separate parts, e.g., mitochondria, lysosomes, etc.Time Factors: Elements of limited time intervals, contributing to particular results or situations.

The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. (1/2135)

When mammalian cells are subjected to calcium depletion stress or protein glycosylation block, the transcription of a family of glucose-regulated protein (GRP) genes encoding endoplasmic reticulum (ER) chaperones is induced to high levels. The consensus mammalian ER stress response element (ERSE) conserved among grp promoters consists of a tripartite structure CCAAT(N9)CCACG, with N being a strikingly GC-rich region of 9 bp. The ERSE, in duplicate copies, can confer full stress inducibility to a heterologous promoter in a sequence-specific but orientation-independent manner. In addition to CBF/NF-Y and YY1 binding to the CCAAT and CCACG motifs, respectively, we further discovered that an ER stress-inducible complex (ERSF) from HeLa nuclear extract binds specifically to the ERSE. Strikingly, the interaction of the ERSF with the ERSE requires a conserved GGC motif within the 9 bp region. Since mutation of the GGC triplet sequence also results in loss of stress inducibility, specific sequence within the 9 bp region is an integral part of the tripartite structure. Finally, correlation of factor binding with stress inducibility reveals that ERSF binding to the ERSE alone is not sufficient; full stress inducibility requires integrity of the CCAAT, GGC and CCACG sequence motifs, as well as precise spacing among these sites.  (+info)

Mechanisms involved in the metabotropic glutamate receptor-enhancement of NMDA-mediated motoneurone responses in frog spinal cord. (2/2135)

1. The metabotropic glutamate receptor (mGluR) agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10-100 microM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 microM). 2. In the presence of NMDA open channel blockers [Mg2+; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801); 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] and TTX, trans-ACPD significantly potentiated NMDA-induced motoneurone depolarizations, but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)- or kainate-induced depolarizations. 3. NMDA potentiation was blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (240 microM), but not by alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (MCCG) (290 microM) or by alpha-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 microM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 microM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 microM). Therefore, trans-ACPD's facilitatory effects appear to involve group I mGluRs. 4. Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3-6 ng ml(-1), 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 microM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCI (H9) (77 microM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 microM) had no effect. 5. Intracellular Ca2+ depletion with thapsigargin (0.1 microM) (which inhibits Ca2+/ATPase), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 microM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+-free medium all reduced trans-ACPD's effects. 6. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 microM) and chlorpromazine (100 microM) diminished the potentiation. 7. In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+-produced channel block of the NMDA receptor.  (+info)

Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel. (3/2135)

Capacitative Ca2+ entry (CCE) is Ca2+ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca2+ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca2+ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca2+-permeable cation channels activated by agonists that stimulate IP3 formation-very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca2+ entry channel. The role of Trp's in Ca2+ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of Ca2+ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca2+ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca2+ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca2+ channels.  (+info)

Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body. (4/2135)

We have previously shown that stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin (0.1 micrometer), an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. Our findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers.  (+info)

Isosmotic modulation of Ca2+-regulated exocytosis in guinea-pig antral mucous cells: role of cell volume. (5/2135)

1. Exocytotic events and changes of cell volume in mucous cells from guinea-pig antrum were examined by video-enhanced optical microscopy. 2. Acetylcholine (ACh) evoked exocytotic events following cell shrinkage, the frequency and extent of which depended on the ACh concentration. ACh actions were mimicked by ionomycin and thapsigargin, and inhibited by Ca2+-free solution and Ca2+ channel blockers (Ni2+, Cd2+ and nifedipine). Application of 100 microM W-7, a calmodulin inhibitor, also inhibited the ACh-induced exocytotic events. These results indicate that ACh actions are mediated by intracellular Ca2+ concentration ([Ca2+]i) in antral mucous cells. 3. The effects of ion channel blockers on exocytotic events and cell shrinkage evoked by ACh were examined. Inhibition of KCl release (quinine, Ba2+, NPPB or KCl solution) suppressed both the exocytotic events and cell shrinkage evoked by ACh. 4. Bumetanide (inhibition of NaCl entry) or Cl--free solution (increasing Cl- release and inhibition of NaCl entry) evoked exocytotic events following cell shrinkage in unstimulated antral mucous cells and caused further cell shrinkage and increases in the frequency of exocytotic events in ACh-stimulated cells. However, Cl--free solution did not evoke exocytotic events in unstimulated cells in the absence of extracellular Ca2+, although cell shrinkage occurred. 5. To examine the effects of cell volume on ACh-evoked exocytosis, the cell volume was altered by increasing the extracellular K+ concentration. The results showed that cell shrinkage increases the frequency of ACh-evoked exocytotic events and cell swelling decreases them. 6. Osmotic shrinkage or swelling caused the frequency of ACh-evoked exocytotic events to increase. This suggests that the effects of cell volume on ACh-evoked exocytosis under anisosmotic conditions may not be the same as those under isosmotic conditions. 7. In antral mucous cells, Ca2+-regulated exocytosis is modulated by cell shrinkage under isosmotic conditions.  (+info)

Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3-sensitive Ca2+ store in guinea-pig colonic smooth muscle. (6/2135)

1. Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+]c) in guinea-pig single colonic myocytes has been examined, using whole-cell recording, flash photolysis of caged InsP3 and microfluorimetry. 2. Depolarization increased [Ca2+]c and triggered contraction. Resting [Ca2+]c was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50 % of its fully relaxed length. The muscle then slowly relaxed (t = 17 s). 3. The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+]c. The extent of the overshoot or undershoot increased with increasing peak [Ca2+]c. 4. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 microM), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high ( > 300 nM) but not at lower [Ca2+]c and abolished [Ca2+]c overshoots. Oligomycin B (5 microM), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5. During depolarization, the global rhod-2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+]m) rose slowly in a CCCP-sensitive manner during and for about 3 s after depolarization had ended. [Ca2+]m then slowly decreased over tens of seconds. 6. Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7. Flash photolysis of caged InsP3 (20 microM) evoked reproducible increases in [Ca2+]c. CCCP (5 microM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 microM) did not reduce the inhibition of the InsP3-induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8. While CCCP reduced the magnitude of the InsP3-evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin-evoked Ca2+ release, did not decrease significantly. 9. [Ca2+]c decline in smooth muscle, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3-evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.  (+info)

Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens. (7/2135)

1. An increase in lens cell calcium has long been associated with cortical cataract. Recently, it has been shown that thapsigargin induces a rise in lens cell calcium by release from endoplasmic reticulum stores. The effects of this rise on the optical and membrane characteristics of the lens were studied in the isolated rat lens. 2. The electrical characteristics of the isolated, perifused rat lens were measured using a two-internal microelectrode technique that permits measurement of plasma membrane conductance (Gm), membrane potential (Vm) and junctional conductance in the intact lens. 3. Thapsigargin (1 microM) induced a rapid overall depolarization of Vm that was accompanied by first a decrease and then an increase in Gm. 4. Replacing external Na+ with tetraethylammonium (TEA) abolished the decrease in Gm. However, a transient increase phase was still observed. 5. The changes in conductance were further characterized by measuring 22Na+ and 45Ca2+ influxes into the isolated lens. Thapsigargin (1 microM) induced a transient increase in 45Ca2+, but did not affect Na+ influx. 6. The Ca2+ channel blocker La3+ (10 microM) totally inhibited the thapsigargin-induced Ca2+ influx. It also blocked the increase in Gm observed in control and in Na+-free-TEA medium. In the absence of external calcium, thapsigargin induced a small depolarization in Vm. 7. These data indicate that thapsigargin induces both a decrease in K+ conductance and an increase in Ca2+ conductance. These probably result from release of stored Ca2+ and subsequent activation of store-operated Ca2+ channels (capacitative Ca2+ entry). 8. Thapsigargin application over the time course of these experiments (24 h) had no effect on junctional conductance or on the transparency of the lens.  (+info)

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture. (8/2135)

Transduction mechanisms between target cells within the intestinal wall and peripheral terminals of extrinsic primary afferent neurons are poorly understood. The purpose of this study was to characterize the interactions between smooth muscle cells from the rat distal colon and lumbar dorsal root ganglion (DRG) neurons in coculture. DRG neurons visually appeared to make contact with several myocytes. We show that brief mechanical stimulation of these myocytes resulted in intracellular Ca2+ concentration ([Ca2+]i) transients that propagated into 57% of the contacting neurites. Direct mechanical stimulation of DRG neurites cultured without smooth muscle had no effect. We also show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins formed functional gap junctions, as evidenced by the intercellular transfer of Lucifer yellow. Furthermore, thapsigargin pretreatment and neuronal heparin injection abolished the increase in neurite [Ca2+]i, indicating that the neuronal Ca2+ signal was triggered by inositol 1,4, 5-trisphosphate-mediated Ca2+ release from intracellular stores. Our results provide evidence for intercellular chemical communication between DRG neurites and intestinal smooth muscle cells that mediates the exchange of second messenger molecules between different cell types.  (+info)

  • Thapsigargin inhibits Ca 2+ -transporting ATPase mediated uptake of calcium ions into sarcoplasmic reticulum and is used in experimentation examining the impacts of increasing cytosolic calcium concentrations. (
  • This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. (
  • Thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, is an important tool in inducing store-regulated Ca2+ influx. (
  • Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. (
  • Intravesicular 45 Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca 2+ -ATPase inhibitor thapsigargin. (
  • The sarcoendoplasmic Ca 2+ -ATPase inhibitors thapsigargin and cyclopiazonic acid did not affect the PACAP-induced sustained rise of [Ca 2+ ] i , but did inhibit the initial [Ca 2+ ] i rise. (
  • The calcium release induced by thapsigargin (a Ca 2+ -ATPase inhibitor) was lower after puromycin treatment. (
  • Increased [Ca 2+ ] i is lowered by reuptake via the thapsigargin (Tg)-sensitive sarcoplasmic Ca 2+ -ATPase and extrusion via the Na + -Ca 2+ exchanger as well as the sarcolemmal Ca 2+ -ATPase ( Bers, 1997 ). (
  • This contraction was also inhibited by ethacrynic acid, by inhibitors of Na+, K+-ATPase (ouabain), Ca2+- ATPase (thapsigargin), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by a non-selective phosphodiesterase inhibitor (papaverine), and by adenosine A2 (metrifudil), and cannabinoid receptor (CP-55940) agonists. (
  • Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs. (
  • The highly oxygenated guaianolide thapsigargin ( 28 ), isolated from the deadly carrot Thapsia garganica , is a selective subnanomolar inhibitor of intracellular calcium ion transport enzymes. (
  • Thapsigargin is derived from the plant Thapsia garganica that produces tumor promoters. (
  • Although knowledge of the efficacy of this plant in the ancient botanical pharmacopoeia was widespread, it was not until recently that the biologically active components of Thapsia were elucidated and determined to be thapsigargin 1, and 15 closely related guaianolides, collectively termed "thapsigargins" (Table 1 and refs. (
  • Among this family of natural compounds, thapsigargin and nortrilobolide are present in the species Thapsia garganica and Thapsia gymnesica [ 2 ]. (
  • This milestone signifies the advancement in Phyton's Plant Cell Fermentation development program that now has begun to define the process by which the Thapsia plant is converted into a preserved, fermentable cell line, thus providing a more sustainable source of high-quality thapsigargin. (
  • Phyton is converting the Thapsia plant into thapsigargin in partnership with Inspyr Therapeutics, Inc. , a clinical-stage biotechnology company that has a patented technology platform utilizing thapsigargin for its prodrug delivery system, mipsagargin. (
  • The chemical compound thapsigargin has been isolated from Thapsia garganica. (
  • In the present paper we show that, at concentrations above 500 nM, thapsigargin also has an opposite effect: it inhibits store-regulated Ca2+ influx into Fura-2-loaded human neutrophil granulocytes. (
  • It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. (
  • These results suggest that emptying of Ca 2+ from intracellular thapsigargin-sensitive Ca 2+ -pumping pools induces profound alteration of cell proliferation. (
  • Here, we demonstrate that murine embryonic fibroblasts deficient for both Bak and Bax are, however, efficiently killed by thapsigargin, a specific inhibitor of ER Ca 2+ pumps that induces ER stress by depleting ER Ca 2+ stores. (
  • Moreover, in cells deficient for both Bak and Bax, thapsigargin induces permanent mitochondrial damage by Ca 2+ overload, permeability transition and membrane rupture. (
  • Importantly, thapsigargin induces caspase-independent cell death also in colon and prostate carcinoma cells deficient in Bak and Bax expression. (
  • Puromycin induces a calcium leak from the ER and decreases the thapsigargin response. (
  • In vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). (
  • Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. (
  • Shoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs. (
  • Thapsigargin is useful in experimentation examining the impacts of increasing cytosolic calcium concentrations. (
  • The cells were incubated at 37°C for 1h in media containing different concentrations of ab120286 (thapsigargin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. (
  • As thapsigargin has been frequently applied at concentrations up to 2 microM, its inhibitory action on plasma-membrane Ca2+ fluxes deserves consideration. (
  • Thapsigargin concentrations as low as 0.1 µM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. (
  • In this study, the influence of external calcium concentrations and thapsigargin on the phagocytosis of polystyrene latex beads by the macrophage-like cell lines MH-S (murine) and differentiated U937 (human) was analyzed. (
  • This process activates thapsigargin, triggering apoptosis of cancer cells. (
  • Due to its ability to kill mammalian cells, thapsigargin has been used as a therapeutic target to induce apoptosis in cancer cells with a low rate of cell proliferation, thus with resistance to standard anti-proliferative chemotherapy. (
  • In the presence of Bak and Bax, thapsigargin eliminates cells by release of mitochondrial cytochrome c and subsequent caspase activation, which leads to the proteolytic inactivation of the molecular necrosis switch PARP-1 and results in apoptosis. (
  • Phospholipase A(2) is involved in thapsigargin-induced sodium influx in human lymphocytes. (
  • Previously, we reported that emptying of intracellular Ca(2+) pools with endoplasmatic Ca(2+)-ATP-ase inhibitor thapsigargin leads to the Na(+) influx in human lymphocytes (M. Tepel et al. (
  • A store-operated Ca2+ influx activated in response to the depletion of thapsigargin-sensitive Ca2+ stores is developmentally regulated in embryonic cortical neurons from mice. (
  • This indicates caffeine-induced hyperactivation depends on external Ca 2 influx, whereas hyperactivation by thapsigargin and thimerosal do not. (
  • To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. (
  • 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. (
  • Ca 2+ measurements with thapsigargin-induced Ca 2+ -depletion/Ca 2+ -restoration protocol to estimate ROCE showed that the stimulation of ET A R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. (
  • Jiang, Xuejun "Distinct Autophagosomal-Lysosomal Fusion Mechanism Revealed by Thapsigargin-Induced Autophagy Arrest" Molecular cell doi:10.1016/j.molcel.2011.04.024 (volume 42 issue 6 pp.731 - 743) Drew, D.P. (
  • Thapsigargin also interferes with mechanisms of autophagy. (
  • Autophagy was initially characterized in candida as an activity utilized by cells to buy Thapsigargin survive metabolic tension (59). (
  • Oddly enough, a latest work demonstrated that E2F1 binds the promoters, raising their buy Thapsigargin appearance and regulating autophagy (44). (
  • We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. (
  • Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. (
  • Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. (
  • In this study, we found that thapsigargin-induced ER stress augmented recruitment of IFN regulatory factor-3, CREB binding protein/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS stimulation. (
  • ab58668 staining ATF3 in serum starved A549 cells treated with thapsigargin (ab120286), by ICC/IF. (
  • A key feature with thapsigargin is that it kills both slow and fast-growth cancer cells by inhibiting an enzyme that controls essential calcium balance inside cells. (
  • In the presence or absence of external calcium ions, thapsigargin-induced calcium signals were transferred to the nucleus as well as to the cytoplasm of RBL-2H3 cells. (
  • Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. (
  • Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. (
  • A23187- and thapsigargin-dependent increase in calreticulin mRNA levels (A) and protein levels (B). (A) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or DMSO. (
  • lane 3) thapsigargin-treated cells. (
  • B) NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin and cellular extracts were prepared as described by Mery et al. (
  • At E13, the release of Ca2+ from thapsigargin-sensitive compartments gave rise to an entry of Ca2+ in a minority of cells. (
  • After only a brief (30-min) treatment of cultured cells with 3 μM thapsigargin followed by extensive washing, the total releasable InsP 3 -sensitive Ca 2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. (
  • Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. (
  • Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca 2+ levels. (
  • Furthermore, the presence of thapsigargin, known to lead to an increase of cytosolic calcium levels, did not have a significant enhancing influence on bead uptake by MH-S cells and only an enhancing effect on bead uptake by macrophage-like U937 cells at an external calcium concentration of 4 mM. (
  • A biotech company called GenSpera, Inc. in San Antonio, TX is studying methods of delivering thapsigargin directly to cancer cells, avoiding damage to other cells in the body of the patient. (
  • Breast carcinoma MCF-7, MDA-MB-231, HS578T and HCC1500 cells were treated with three different ER stressors - Brefeldin A, Tunicamycin, Thapsigargin - and levels of PrP mRNA or protein assessed by RT-PCR and Western blot analyses. (
  • Additionally, the role of calcium signalling and PKC activation on facilitative glucose transport was examined by measuring glucose uptake in BBM vesicles prepared from proximal tubules that had been incubated either with thapsigargin, which increases cytosolic calcium, or with the PKC activator phorbol 12-myristate,13-acetate (PMA). (
  • Thapsigargin and PMA enhanced GLUT-mediated glucose uptake, but had no effect on sodium-dependent glucose transport. (
  • 2) In epithelium-intact smooth muscle, thapsigargin significantly suppressed the contraction evoked by acetylcholine, suggesting that thapsigargin stimulate the epithelium to produce EpDRF. (
  • It is not known whether the secondary modifications to the guaianolide occur before, or after the formation of thapsigargin, but will need to be considered when elucidating the true biosynthesis. (