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The role of nitric oxide in cytoskeleton-mediated organelle transport and cell adhesion
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nitric oxide (NO) is a signaling molecule that is produced by many different kinds of cells, and it is known to mediate actions such as blood vessel dilation, communication between nerve cells, and killing of bacteria in infections. The cytoskeleton is involved in many important cellular functions, among them intracellular transport of organelles, migration, and cell division. The aim of the present studies was to examine the effects of NO on some of the indicated functions. Homotypic adhesion of human neutrophils, which is mediated by ß2 integrins, is an early step in the inflammatory process. Addition of L-argiriine (the substrate of NO production) to fMLP-stimulated neutrophils increased and prolonged aggregation of the cells. Stimulation of L-arginine-pretreated neutrophils by cross-linking of ß2 integrins attenuated the increase in F-actin, as compared to control cells. These results suggest that the aggregation is prolonged by activation of ß2 integrins and endogenous NO production, two events that together seem to inhibit actin polymerization, possibly via ADP ribosylation.

The effect of NO on intracellular translocation of organelles along the cytoskeleton was studied in Xenopus laevis pigment cells. Inhibition of NO production induced by the drug L-NAME was found to inhibit aggregation of the pigment organelles (melanosomes) and to induce dispersion. Activation of PKC, MEK, and ERKl, but not PKA, was associated with the dispersion, thus NO may negatively regulate these kinases, which, when activated, would induce movement of melanosomes. During melanosome aggregation, the cell center increases in height by approximately 30%. Experiments were performed to determine whether the cell membrane is pushed upwards by actin polymerization and water influx through HgCl2-sensitive aquaporins. The results gave no evidence that either two of these mechanisms affects the upward movement. However, L-NAME caused dispersion and a decrease in cell height, thus NO may play a role in maintaining an aggregated, elevated state. In conclusion, many factors regulate both homotypic aggregation and intracellular organelle transport, and NO seems to prolong homotypic aggregation of neutrophils and regulate melanosome transport by inhibiting PKC, MEK and ERKl.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 48 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 660
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28614Local ID: 13769ISBN: 91-7219-761-7 (print)OAI: oai:DiVA.org:liu-28614DiVA: diva2:249425
Public defence
2001-03-02, Elsa Brändströmssalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2015-09-18Bibliographically approved
List of papers
1. Nitric Oxide Regulates the Aggregation of Stimulated Human Neutrophils
Open this publication in new window or tab >>Nitric Oxide Regulates the Aggregation of Stimulated Human Neutrophils
2000 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 274, no 2, 482-487 p.Article in journal (Refereed) Published
Abstract [en]

Neutrophil aggregation is mediated by both CD18 integrin and L-selectin. Nitric oxide attenuates the integrin-mediated adhesion of neutrophils to collagen and to endothelium and may therefore affect aggregation as well. FMLP-stimulated neutrophils exposed to -arginine showed increased and prolonged aggregation, whereas cells pretreated with L-NAME did not differ from FMLP-stimulated controls. Nitric oxide is known to induce ADP ribosylation of G-actin, which inhibits polymerization. We detected equivalent levels of total F-actin in cells pretreated with -arginine or L-NAME and non-pretreated controls. However, neutrophils pretreated with -arginine and stimulated by CD18 integrin cross-linking exhibited a more limited increase in total F-actin, compared to control and L-NAME-pretreated cells. Thus at least two signaling pathways may be involved FMLP-stimulated aggregation, mediated by CD18 integrins. More specifically, it is plausible that FMLP-receptor signaling upregulates CD18 integrins and endogenous NO subsequently modulates CD18-mediated signaling to prolong aggregation, possibly through ADP-ribosylation of actin.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25912 (URN)10.1006/bbrc.2000.3156 (DOI)10354 (Local ID)10354 (Archive number)10354 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2015-09-18Bibliographically approved
2. Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores
Open this publication in new window or tab >>Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores
Show others...
2000 (English)In: Cell Motility and the Cytoskeleton, ISSN 0886-1544, E-ISSN 1097-0169, Vol. 47, no 3, 209-218 p.Article in journal (Refereed) Published
Abstract [en]

Pigment organelles in Xenopus laevis melanophores are used by the animal to change skin color, and they provide a good model for studying intracellular organelle transport. Movement of organelles and vesicles along the cytoskeleton is essential for many processes, such as axonal transport, endocytosis, and intercompartmental trafficking. Nitric oxide (NO) is a signaling molecule that plays a role in, among other things, relaxation of blood vessels, sperm motility, and polymerization of actin. Our study focused on the effect NO exerts on cytoskeleton-mediated transport, which has previously received little attention. We found that an inhibitor of NO synthesis, N-nitro-L-arginine methyl ester (L-NAME), reduced the melatonin-induced aggregation of the pigment organelles, melanosomes. Preaggregated melanosomes dispersed after treatment with L-NAME but not after exposure to the inactive stereoisomer (D-NAME) or the substrate for NO synthesis (L-arginine). Signal transduction by NO can be mediated through the activation of soluble guanylate cyclase (sGC), which leads to increased production of cGMP and activation of cGMP-dependent kinases (PKG). We found that both the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the cGMP analogue 8-bromoguanosine 3′:5′-cyclic monophosphate (8-Br-cGMP) reduced melanosome aggregation, whereas the PKG inhibitor KT582 did not. Our results demonstrate that melanosome aggregation depends on synthesis of NO, and NO deprivation causes dispersion. It seems, thus, as if NO and cGMP are essential and can regulate melanosome translocation.

Keyword
melanosome, aggregation, cGMP, microtubules, actin, L-NAME
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13570 (URN)10.1002/1097-0169(200011)47:3<209::AID-CM4>3.0.CO;2-W (DOI)
Available from: 2001-03-14 Created: 2001-03-14 Last updated: 2015-09-18Bibliographically approved
3. L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1
Open this publication in new window or tab >>L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1
2001 (English)In: Pigment Cell Research, ISSN 0893-5785, E-ISSN 1755-148X, Vol. 14, no 6, 450-455 p.Article in journal (Refereed) Published
Abstract [en]

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω-nitro-l-arginine methyl ester (l-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l-NAME-dispersed melanophores. l-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26064 (URN)10.1034/j.1600-0749.2001.140605.x (DOI)10523 (Local ID)10523 (Archive number)10523 (OAI)
Note

On the day of the defence day the status of this article was a manuscript.

Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2015-09-18Bibliographically approved
4. HgCl2-sensitive aquaporins are not involved in melanosome aggregation in Xenopus laevis melanophores
Open this publication in new window or tab >>HgCl2-sensitive aquaporins are not involved in melanosome aggregation in Xenopus laevis melanophores
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Melanophores are cells specialized for transport of pigment-filled organelles called melanosomes. Melanosomes are aggregated in the center of a melanophore or dispersed throughout the cytoplasm by motor proteins moving along the actin and microtubule cytoskeleton. In angelfish (Pterophyllum scalare), aggregation of melanosomes (as compared to dispersion) increases the height of the central part of melanophores by 300%. Our objective was to detennine whether such a height increase also occurs in frog (Xenopus laevis) melanophores. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that elevation of the melanophore plasma membrane is due to local swelling caused by influx of water through HgCl2-sensitive aquaporins and subsequent polymerization of actin. Confocal microscopy revealed a 30% increase in height in X. laevis melanophores during melatonin-induced aggregation. This was not due to actin polymerization, because it also occurred when aggregation was induced by the polymerization inhibitor latrunculin B. The nitric oxide (NO) synthase inhibitor L-NAME induced dispersion and lowered the plasma membrane, which suggests that NO is involved in the upward movement. Furthermore, neither dispersion nor aggregation was affected by inhibition of water flux through HgCl2 sensitive aquaporins. Together, these observations imply that melanosomes in X. laevis melanophores are driven upwards during aggregation by a mechanism other than actin polymerization, possibly involving microtubules, intermediate filaments, or a motor protein that may be regulated by NO. Furthermore, influx of water through HgCl2-sensitive aquaporins is probably not necessary for aggregation-induced elevation of the cell membrane, because both aggregation and dispersion can occur in the presence of HgCl2.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80030 (URN)
Available from: 2012-08-17 Created: 2012-08-17 Last updated: 2015-09-18Bibliographically approved

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