Nitric oxide induces dose-dependent CA2+ transients and causes temporal morphological hyperpolarization in human neutrophils

Abstract

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.

The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity

“…Caco-2 cells were cultured on 35-mm glass-bottom culture dishes (MatTek, Ashland, MA) to 70 to 80% confluence and maintained in fresh DMEM overnight before use. After being washed with PBS (pH 7.6), the cells were loaded along with 8 M Fura- (23). Image Master 5.0 software (Photon Technology, Birmingham, NJ) was used for the control of the instrument and data acquisition.…”

Nitric Oxide in Astrocyte-Neuron Signaling

“…The three ATP application protocol was also used with the first and third ATP applications being administrated in normal saline whereas the second ATP application was carried out after PPADS treatment. direct nitrosylation of proteins (Willmott et al, 1995;Guihard et al, 1996;Rooney et al, 1996;Willmott et al, 1996;Lee, 1997;Volk et al, 1997;Berkels et al, 2000;Loitto et al,, 2000;Looms et al, 2001), but its roles in astrocytes are largely unknown. 2+ .…”

“…The cGMP-G Kinase can also result in phosphorylation of I P 3 receptors (Guihard et al, 1996;Rooney et al, 1996). NO has also been demonstrated to liberate Ca2' by a cGMP independent pathway (VoIk et al, 1997;Watson et al, 1999;Berkels et a]., 2000;Loitto et al, 2000). All these findings raise the possibility that NO may also induce Ca2+ mobilization in astrocytes, and in turn modulate Ca"-dependent glutamate release from astrocyte to neuron.…”

Nitric oxide triggers specific and dose-dependent cytosolic calcium transients in Arabidopsis

“…28 Thus, SNP has been previously used as a potential NO donor to study NO-mediated signaling in mammalian and plant systems. 17,29,30 Therefore, SNP was utilized as the NO-donor in all experiments. Previously, luminescence response has been successfully used as a satisfactory parameter for qualitative analysis of changes in [Ca 2+ ] cyt in response to oxidative signals.…”

“…It has been demonstrated the ability of sodium nitroprusside (SNP), an NO donor, and to elicit dose-dependent Ca 2+ fluxes in human neutrophiles. 17 Moreover, it has been indicated that exogenous NO donor SNP induced the release of Ca 2+ from intracellular IP(3) receptorsensitive stores of respiratory burst neutrophils via S-nitrosylation. 18 To this end, NO was found to play a crucial role in plant growth and development, starting from germination to flowering, ripening of fruit and senescence of organs.…”