thapsigargin and tetrafluoroaluminate

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.

Topics: Aluminum Compounds; Amino Acid Sequence; Blood Platelets; Calcium; Cell Line; Cell Membrane; Egtazic Acid; Endoplasmic Reticulum; Fluorides; Humans; Hydroquinones; Isoenzymes; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Trypsin

To evaluate the possible mechanisms responsible for the anti-inflammatory effects of baicalin or baicalein, phorbol-12-myristate-13-acetate (PMA)- or N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated inflammatory responses of peripheral human leukocytes were studied. Both baicalin and baicalein diminished fMLP- or PMA-induced reactive oxygen intermediates production in neutrophils or monocytes. Neither baicalin nor baicalein prevented the protein kinase C (PKC)-dependent assembly of the NADPH oxidase. Conversely, myeloperoxidase (MPO) activity was inhibited by baicalin or baicalein. fMLP-induced activation of leukocytes, as reflected by increased surface expression of Mac-1 (CD11b/CD18) and Mac-1-dependent neutrophil adhesion, were also inhibited by baicalin or baicalein. Furthermore, baicalein, but not baicalin, impeded fMLP- or AlF(4)(-)-induced Ca(2+) influx. We conclude that impairment of reactive oxygen intermediates production, through scavenging reactive oxygen intermediates by baicalin, or antagonizing ligand-initiated Ca(2+) influx by baicalein, accounts for the inhibition of Mac-1-dependent leukocyte adhesion that confers the anti-inflammatory activity of baicalin or baicalein.

Topics: Adult; Aluminum Compounds; Anti-Infective Agents; Calcium; Cell Adhesion; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Flavanones; Flavonoids; Fluorides; Free Radical Scavengers; Humans; Leukocytes; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Peroxidase; Protein Kinase C; Reactive Oxygen Species; Thapsigargin

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and AlF4(-) inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3',5'-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

Topics: Adenosine Triphosphate; Aluminum Compounds; Animals; Bucladesine; Calcium; Calcium Channels; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Enzyme Inhibitors; Fluorides; Genistein; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Liver; Mitochondria, Liver; Rats; Thapsigargin; Vasopressins

Calcium signaling within astrocytes in the CNS may play a role comparable to that of electrical signaling within neurons. ATP is a molecule known to produce Ca2+ responses in astrocytes, and has been implicated as a mediator of intercellular Ca2+ signaling in other types of nonexcitable cells. We characterized the signal transduction pathway for ATP-evoked Ca2+ responses in cultured astrocytes from the dorsal spinal cord. Nearly 100% of these astrocytes respond to extracellularly applied ATP, which causes release of Ca2+ from an intracellular pool that is sensitive to thapsigargin and insensitive to caffeine. We found that intracellular administration of IP3 also caused release of Ca2+ from a thapsigargin-sensitive intracellular pool, and that IP3 abolished the response to ATP. The ATP-evoked Ca2+ response was blocked by the IP3 receptor antagonist heparin, applied intracellularly, but not by N-desulfated heparin, which is not an antagonist at these receptors. The Ca2+ response caused by ATP was also blocked by a phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. Increases in [Ca2+]i were elicited by intracellular application of activators of heterotrimeric G-proteins, GTP gamma S and AIF4-. On the other hand, [Ca2+], was unaffected by a G-protein inhibitor, GDP beta S, but it did abolish the Ca2+ response to ATP. Pretreating the cultures with pertussis toxin did not affect responses to ATP. Our results indicate that in astrocytes ATP-evoked release of intracellular Ca2+ is mediated by IP3 produced as a result of activating phospholipase C coupled to ATP receptors via a G-protein that is insensitive to pertussis toxin. ATP is known to be released under physiological and pathological circumstances, and therefore signaling via the PLC-IP3 pathway in astrocytes is a potentially important mechanism by which ATP may play a role in CNS function.

Topics: Adenosine Triphosphate; Aluminum Compounds; Animals; Astrocytes; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Cells, Cultured; Egtazic Acid; Estrenes; Fetus; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Isoenzymes; Patch-Clamp Techniques; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Spinal Cord; Terpenes; Thapsigargin; Thionucleotides; Time Factors; Type C Phospholipases

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).

Topics: 3T3 Cells; Aluminum; Aluminum Compounds; Animals; Brefeldin A; Carrier Proteins; Cell Hypoxia; Cycloheximide; Cyclopentanes; Endoplasmic Reticulum Chaperone BiP; Ethers, Cyclic; Fluorides; Fluorine; Genistein; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Isoflavones; Membrane Proteins; Mice; Molecular Chaperones; Okadaic Acid; Protein Biosynthesis; RNA, Messenger; Terpenes; Thapsigargin