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Nitric oxide induces dose-dependent CA2+ transients and causes temporal morphological hyperpolarization in human neutrophils
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2000 (English)In: Journal of cellular physiology, ISSN 0021-9541, Vol. 182, no 3, 402-413 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2000. Vol. 182, no 3, 402-413 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13604DOI: 10.1002/(SICI)1097-4652(200003)182:3<402::AID-JCP11>3.0.CO;2-DOAI: oai:DiVA.org:liu-13604DiVA: diva2:21044
Available from: 2001-05-25 Created: 2001-05-25 Last updated: 2015-09-18
In thesis
1. Towards a Refined Model of Neutrophil Motility
Open this publication in new window or tab >>Towards a Refined Model of Neutrophil Motility
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2001. 135 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 670
Keyword
human polymorphonuclear leukocytes. PMNL, neutrophils, neutrophil motility, N-formyl-Met-Leu-Phe
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-5142 (URN)91-7219-964-4 (ISBN)
Public defence
2001-05-04, Aulan, Adm. byggnad, ingång 16, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2001-05-25 Created: 2001-05-25 Last updated: 2012-01-24Bibliographically approved

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