calcimycin and tetrafluoroaluminate

Plants can acquire freezing tolerance in response to cold but non-freezing temperatures. To efficiently activate this cold acclimation, low temperature has to be sensed and processed swiftly, a process that is linked with a transient elimination of microtubules. Here, we address cold-induced microtubules elimination in a grapevine cell line stably expressing a green fluorescent protein fusion of Arabidopsis TuB6, which allows to follow their response in vivo and to quantify this response by quantitative image analysis. We use time-course studies with several specific pharmacological inhibitors and activators to dissect the signalling events acting upstream of microtubules elimination. We find that microtubules disappear within 30 min after the onset of cold stress. We provide evidence for roles of calcium influx, membrane rigidification, and activation of NAD(P)H oxidase as factors in signal susception and amplification. We further conclude that a G-protein in concert with a phospholipase D convey the signal towards microtubules, whereas calmodulin seems to be not involved. Moreover, activation of jasmonate pathway in response to cold is required for an efficient microtubule response. We summarize our findings in a working model on a complex signalling hub at the membrane-cytoskeleton interphase that assembles the susception, perception and early transduction of cold signals.

Topics: Aluminum Compounds; Benzyl Alcohol; Biphenyl Compounds; Calcimycin; Calcium; Cell Membrane; Cold Temperature; Cyclopentanes; Cytoplasm; Dimethyl Sulfoxide; Egtazic Acid; Fluorides; Gadolinium; Ionophores; Microtubules; NADPH Oxidases; Nitroprusside; Onium Compounds; Oxylipins; Pertussis Toxin; Phospholipase D; Polymerization; Pyrazoles; Signal Transduction; Stress, Physiological; Vitis

We compared the regulation of cytosolic phospholipase A2 (cPLA2) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca(2+)-mobilizing P2-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P2-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA2 and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca2+, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA2. The stimulatory effects of GTP gamma S and Ca2+ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA2 coupling. Basal and PMA-stimulated phosphorylation of cPLA2 was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA2 phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.

Topics: Adenosine Triphosphate; Aluminum Compounds; Arachidonic Acid; Blotting, Western; Bucladesine; Calcimycin; Calcium; Cell Differentiation; Cell Line; Cytosol; Electrophoresis, Polyacrylamide Gel; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Leukemia, Promyelocytic, Acute; Neutrophils; Phospholipases A; Phospholipases A2; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content of unstimulated human red blood cells (RBC) has been found to be 227 +/- 83 pmol of Ins(1,4,5)P3 per mL of packed cells. Ins (1,4,5)P3 at concentrations between 0.4-5.0 microM releases Ca2+ from RBC plasma membrane with an EC50 of 0.55 microM. Inositol 4,5-bisphosphate [Ins(4,5)P2] is less potent, but inositol 1-phosphate [Ins(1)P], inositol 1,4-bisphosphate [Ins (1,4)P2], and adenosine triphosphate (ATP) are inactive. The release was stereospecific for D-Ins(1,4,5)P3; 1 microM L-Ins(1,4,5)P3 released no more Ca2+ than the control. A nonhydrolyzable analog of Ins(1,4,5)P3, inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)PS3] evokes sustained release of Ca2+ from isolated ghosts. Release of 45Ca2+ was also observed after the addition of AlF4-. Ionophore A23187 and AlF4- increase the level of Ins(1,4,5)P3 in intact RBC to 1004 +/- 533 and 455 +/- 74 pmol/mL of packed RBC, respectively. We have elaborated a method for visualization of spectrin by indirect immunofluorescence in smears of RBC. Ins(1,4,5)P3 evokes shape changes in permeabilized RBC and disorganization of the spectrin network. The shape changes are stereospecific for the D-enantiomer, since L-Ins(1,4,5)P3 and other compounds had no effect. Whereas the effect of Ins(1,4,5)P3 was reversible, as was the weaker effect of Ins(4,5)P2, Ins(1,4,5)PS3 evoked irreversible shape changes. Shape changes and spectrin disorganization were also observed after the action of AlF4- and ionophore A23187. We conclude that the phosphoinositide signaling system plays an important role in the shape maintenance of human RBC.

Topics: Aluminum; Aluminum Compounds; Calcimycin; Calcium; Erythrocyte Membrane; Erythrocytes; Fluorescent Antibody Technique; Fluorides; Fluorine; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Organothiophosphorus Compounds; Spectrin

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 6-Ketoprostaglandin F1 alpha; Aluminum; Aluminum Compounds; Arachidonic Acids; Calcimycin; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Epoprostenol; Female; Fluorides; Fluorine; GTP-Binding Proteins; Humans; Inositol Phosphates; Isoquinolines; Kinetics; Pertussis Toxin; Piperazines; Pregnancy; Protein Kinase C; Signal Transduction; Sodium Fluoride; Tetradecanoylphorbol Acetate; Umbilical Veins; Virulence Factors, Bordetella