Nitric oxide induces dose-dependent CA2+ transients and causes temporal morphological hyperpolarization in human neutrophils
✍ Scribed by Vesa-Matti Loitto; Harriet Nilsson; Tommy Sundqvist; Karl-Eric Magnusson
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 285 KB
- Volume
- 182
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
We exposed adherent neutrophils to the nitric oxide (NO)-radical donors Snitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca 2ϩ signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca 2ϩ ([Ca 2ϩ ] 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 M SNAP caused concentration-dependent elevation of [Ca 2ϩ ] i with an optimal effect at 50 M. This was probably induced by NO itself, because no change in [Ca 2ϩ ] i was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (Ն200 M) attenuated the Ca 2ϩ response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO-and fMLP-induced Ca 2ϩ transients were abolished at 800 M 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 2ϩ ] 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 2ϩ 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.