calcimycin and diphenyliodonium

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

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.

Topics: Biphenyl Compounds; Bradykinin; Calcimycin; Calcium; Cell Line; Epidermal Growth Factor; Flavin-Adenine Dinucleotide; Humans; Hydrogen Peroxide; Ionophores; Keratinocytes; Luminescent Measurements; Onium Compounds; Reactive Oxygen Species; Receptors, Cell Surface; Thapsigargin

Primary cultures of rat Kupffer cells liberated significant amounts of prostaglandin (PG) D2, PGE2, and thromboxane (measured as thromboxane B2) when exposed to reoxygenation after 4 h of hypoxia. After a delayed onset, prostanoids were released at high rates for at least 8 h and after that time 700 pmol PGD2, 280 pmol PGE2, and 200 pmol thromboxane per 10(6) cells had been liberated. Unlike prostanoid release, leukotriene B4 production in reoxygenated cell cultures was only twice as much as in aerobic controls. Superoxide dismutase and catalase had no effect on PGD2, PGE2, and thromboxane production, indicating that prostanoid formation was independent of reactive oxygen species generated extracellularly and of cell injury. On the other hand, diphenyliodonium, as well as amiloride, blocked hypoxia-reoxygenation-induced PGD2, PGE2, and thromboxane release. The elevated prostanoid synthesis was preceded by increases in intracellular pH (from 7.23 to 7.38) and in intracellular Ca2+ (from 55 nM to a maximum level of 807 nM). These observations suggest a participation of NADPH oxidase and a related Na(+)-H+ exchange in the enhanced prostanoid synthesis, probably through the induction of an increased intracellular Ca2+ concentration.

Topics: Amiloride; Animals; Anti-Infective Agents; Biphenyl Compounds; Calcimycin; Calcium; Carrier Proteins; Cell Survival; Cells, Cultured; Dinoprostone; Hydrogen-Ion Concentration; Hypoxia; Kupffer Cells; Leukotriene B4; Male; NADH, NADPH Oxidoreductases; NADPH Oxidases; Onium Compounds; Oxidation-Reduction; Oxygen; Prostaglandin D2; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Thromboxane B2