calcimycin and gadolinium-chloride

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

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

Topics: Adenosine Triphosphate; Calcimycin; Calcium; Carbenoxolone; Cells, Cultured; Connexin 43; Connexins; Egtazic Acid; Gadolinium; Histamine; Human Umbilical Vein Endothelial Cells; Humans; Nerve Tissue Proteins; Oligopeptides; Receptor, PAR-1; RNA, Small Interfering; Thrombin

It is well established that calcium-dependent neurotransmitter release and exocytosis can be regulated by altering the cholesterol content of the plasma membrane. We have compared the influence of cholesterol depletion of synaptosomal plasma membrane by 15 mM methyl-beta-cyclodextrin (MCD) treatment on calcium-dependent release of D-[(3)H]aspartate induced by the calcium ionophore A23187 and on calcium-independent release induced by hypertonic shrinking or polyvalent cations. We found that decrease of cholesterol concentration by 9.3% inhibited calcium-dependent release of d-[(3)H]aspartate induced by calcium ionophore A23187 by four times while release induced by 300 microM Gd(3+), 150 mM and 500 mM sucrose remained unchanged. Further we have investigated the influence of MCD on exocytosis monitored by the fluorescent dye, acridine orange. Cholesterol depletion inhibited calcium-dependent exocytosis induced by calcium ionophore A23187 but had virtually no influence on calcium-independent exocytosis induced by hypertonic shrinking or Gd(3+). In summary, we found that the cholesterol content in synaptosomal plasma membrane is important for calcium-dependent exocytosis.

Topics: Acridine Orange; Animals; Aspartic Acid; beta-Cyclodextrins; Brain; Calcimycin; Calcium; Cell Membrane; Cholesterol; Exocytosis; Fluorescent Dyes; Gadolinium; Hypertonic Solutions; In Vitro Techniques; Ionophores; Male; Rats; Rats, Wistar; Synaptosomes