Propofol causes neurite retraction in neurones.

BACKGROUND: The mechanism by which anaesthetic agents produce general anaesthesia is not yet fully understood. Retraction of neurites is an important function of individual neurones and neural plexuses during normal and pathological conditions, and it has been shown that such a retraction pathway exists in developing and mature neurones. We hypothesized that propofol decreases neuronal activity by causing retraction of neuronal neurites. METHODS: Primary cultures of rat cortical neurones were exposed in concentration- and time-response experiments to 0.02, 0.2, 2, and 20 microM propofol or lipid vehicle. Neurones were pretreated with the GABA(A) receptor (GABA(A)R) antagonist, bicuculline, the myosin II ATPase activity inhibitor, blebbistatin, and the F-actin stabilizing agent, phalloidin, followed by administration of propofol (20 microM). Changes in neurite retraction were evaluated using time-lapse light microscopy. RESULTS: Propofol caused a concentration- and time-dependent reversible retraction of cultured cortical neurone neurites. Bicuculline, blebbistatin, and phalloidin completely inhibited propofol-induced neurite retraction. Images of retracted neurites were characterized by a retraction bulb and a thin trailing membrane remnant. CONCLUSIONS: Cultured cortical rat neurones retract their neurites after exposure to propofol in a concentration- and time-dependent manner. This retraction is GABA(A)R mediated, reversible, and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to those observed during propofol anaesthesia.

Assessment of neutrophil N-formyl peptide receptors by using antibodies and fluorescent peptides.

Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.

Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores.

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.

Nitric oxide induces dose-dependent CA(2+) transients and causes temporal morphological hyperpolarization in human neutrophils.

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.