Dual effect of cannabinoid CB<Subscript>1</Subscript> receptor stimulation on a vanilloid VR1 receptor-mediated response

Cannabinoid CB1 receptors and vanilloid VR1 receptors are co-localized to some extent in sensory neurons of the spinal cord and dorsal root ganglia. In this study, we over-expressed both receptor types in human embryonic kidney (HEK)-293 cells and investigated the effect of the CB1 agonist HU-210 on the VR1-mediated increase in intracellular Ca2+ ([Ca2+]i), a well-known response of the prototypical VR1 agonist capsaicin. After a 5-min pre-treatment, HU-210 (0.1 microM) significantly enhanced the effect of several concentrations of capsaicin on [Ca2+]i in HEK-293 cells over-expressing both rat CB1 and human VR1 (CB1-VR1-HEK cells), but not in cells over-expressing only human VR1 (VR1-HEK cells). This effect was blocked by the CB1 receptor antagonist SR141716A (0.5 microM), and by phosphoinositide-3-kinase and phospholipase C inhibitors. The endogenous agonist of CB1 and VR1 receptors, anandamide, was more efficacious in inducing a VR1-mediated stimulation of [Ca2+]i in CB1-VR1-HEK cells than in VR1-HEK cells, and part of its effect on the former cells was blocked by SR141716A (0.5 microM). Pre-treatment of CB1-VR1-HEK cells with forskolin, an adenylate cyclase activator, enhanced the capsaicin effect on [Ca2+]i. HU-210, which in the same cells inhibits forskolin-induced enhancement of cAMP levels, blocked the stimulatory effect of forskolin on capsaicin. Our data suggest that in cells co-expressing both CB1 and VR1 receptors, pre-treatment with CB1 agonists inhibits or stimulates VR1 gating by capsaicin depending on whether or not cAMP-mediated signalling has been concomitantly activated.     

Nociception,neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice

Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice. Thermoregulatory behaviour in relation to environmental thermopreference (30 vs. 35°C) was slightly impaired in male knockdown mice, but the reduction of TRPV1 function was not associated with enhanced hyperthermia. TRPV1 agonist resiniferatoxin induced hypothermia and tail vasodilatation was markedly inhibited in knockdown mice. In conclusion, shRNA-mediated knock down of the TRPV1 receptor in mice induced robust inhibition of the responses to TRPV1 agonists without altering the expression, gating function or neurogenic oedema provoked by TRPA1 activation. Thermoregulatory behaviour in response to heat was inhibited, but enhanced hyperthermia was not observed.     

Signaling mechanisms of neurite outgrowth induced by the cell adhesion molecules NCAM and N-Cadherin

Formation of appropriate neural circuits depends on a complex interplay between extracellular guiding cues and intracellular signaling events that result in alterations of cytoskeletal dynamics and a neurite growth response. Surface-expressed cell adhesion molecules (CAMs) interact with the surroundings via the extracellular domain and bind to the cytoskeleton via their intracellular domain. In addition, several CAMs induce signaling events via direct interactions with intracellular proteins or via interactions with cell surface receptors. Thus, CAMs are obvious candidates for transmitting extracellular guidance cues to intracellular events and thereby regulating neurite outgrowth. In this review, we focus on two CAMs, the neural cell adhesion molecule (NCAM) and N-cadherin, and their ability to mediate signaling associated with a neurite outgrowth response. In particular, we will focus on direct interaction between NCAM and N-cadherin with a number of intracellular partners, as well as on their interaction with the fibroblast growth factor receptor (FGFR). Received 23 May 2008; received after revision 14 July 2008; accepted 21 July 2008     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.Received 26 June 2003; received after revision 29 July 2003; accepted 25 August 2003     

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Gα_q protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.     

Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm

The means by which oxygen intervenes in gene expression has been examined in considerable detail in the metabolically versatile bacterium Rhodobacter sphaeroides. Three regulatory systems are now known in this organism, which are used singly and in combination to modulate genes in response to changing oxygen availability. The outcome of these regulatory events is that the molecular machinery is present for the cell to obtain energy by means that are best suited to prevailing conditions, while at the same time maintaining cellular redox balance. Here, we explore the dangers associated with molecular oxygen relative to the various metabolisms used by R. sphaeroides, and then present the most recent findings regarding the features and operation of each of the three regulatory systems which collectively mediate oxygen control in this organism.Received 26 June 2003; received after revision 30 July 2003; accepted 8 August 2003     

Regulation of macrophage activation

IFN- rapidly primes the macrophage via JAK1/2-STAT1 pathway so that it can subsequently undergo a slower classical type 1 activation upon exposure to T helper (Th)1 cytokines such as IFN or other activators, including tumor necrosis factor and lipopolysaccharide, e.g. in intracellular killing of phagocytosed Mycobacterium tuberculosis. If instead it is driven by Th2 cytokines interleukin (IL)-4 and IL-13, it undergoes alternate type 2 activation, which enhances endocytotic antigen uptake and presentation, mast cell and eosinophil involvement and type 2 granuloma formation, e.g. in response to parasitic and extracellular pathogens. Particle-induced macrophage activation was shown to differ from classical and alternate activation, showing in DNA microarray experiments (complete linkage/ Euclidean distance metric analysis) upregulation of nonsecreted structural/signaling molecules and lack of secreted proin-flammatory cyto- and chemokines. The switch-off (deactivation) of already activated macrophages is an active, controlled process in which IL-10 and corticosteroids play important roles and to which15dPGJ2, PGA1/2 and vasoactive intestinal peptide often contribute.Received 16 January 2003; received after revision 14 March 2003; accepted 2 April 2003     

A novel pattern of fast calcium oscillations points to calcium and electrical activity cross-talk in rat chromaffin cells

Slow oscillations of cytosolic calcium ion concentration – – typically originate from release by intracellular stores, but in some cell types can be triggered and sustained by Ca²⁺ influx as well. In this study we simultaneously monitored changes in and in the electrical activity of the cell membrane by combining indo-1 and patch-clamp measurements in single rat chromaffin cells. By this approach we observed a novel type of spontaneous oscillations, much faster than those previously described in these cells. These oscillations are triggered and sustained by complex electrical activity (slow action potentials and spike bursts), require Ca²⁺ influx and do not involve release from intracellular stores. The possible physiological implications of this new pathway of intracellular signalling are discussed.Received 30 July 2004; received after revision 14 October 2004; accepted 1 November 2004     

Loperamide: novel effects on capacitative calcium influx

Loperamide is a widely used antidiarrheal that primarily acts at nanomolar concentrations through activation of opioid receptors in the gastrointestinal tract. At somewhat higher concentrations, loperamide blocks calmodulin activity, calcium channels, N-methyl-D-aspartate-receptor channels, and maitotoxin-elicited calcium influx. Loperamide at micromolar concentrations has now been shown to have a remarkable stimulatory effect on the capacitative calcium influx that is triggered in many cells by depletion of the inositol-trisphosphate-sensitive stores of calcium in the endoplasmic reticulum. The mechanism whereby loperamide enhances levels of intracellular calcium elevated by capacitative calcium influx is, as yet, undefined.     

Targeting of the Akt/PKB kinase to the actin skeleton

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.Received 10 September 2003; accepted 25 September 2003     

The role of calcium in aggregation and development ofDictyostelium

Changes in cytosolic Ca²⁺ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca²⁺]_i) at a low level in the face of steep gradients of extracellular and sequestered Ca²⁺. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca²⁺ into the cytosol and elevation of [Ca²⁺]_i. After such stimulation Ca²⁺ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca²⁺]_i to its basal level. Such responses to elevation of [Ca²⁺]_i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.     

The complexity of parathyroid hormone-related proteinsignalling

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.Received 19 June 2003; received after revision 10 July 2003; accepted 21 July 2003     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.     

Caffeine as a psychomotor stimulant: mechanism of action

The popularity of caffeine as a psychoactive drug is due to its stimulant properties, which depend on its ability to reduce adenosine transmission in the brain. Adenosine A₁ and A_2A receptors are expressed in the basal ganglia, a group of structures involved in various aspects of motor control. Caffeine acts as an antagonist to both types of receptors. Increasing evidence indicates that the psychomotor stimulant effect of caffeine is generated by affecting a particular group of projection neurons located in the striatum, the main receiving area of the basal ganglia. These cells express high levels of adenosine A_2A receptors, which are involved in various intracellular processes, including the expression of immediate early genes and regulation of the dopamine- and cyclic AMP-regulated 32-kDa phosphoprotein DARPP-32. The present review focuses on the effects of caffeine on striatal signal transduction and on their involvement in caffeine-mediated motor stimulation.Received 8 July 2003; received after revision 7 September 2003; accepted 6 October 2003     

Induced recovery of defective membrane expression of a CC chemokine receptor 5 mutant by phytohemagglutinin

CC chemokine receptor 5 (CCR5) is a member of the G-protein-coupled receptor superfamily. It plays an important role in macrophage tropic human immunodeficiency virus-1 entry and in some inflammatory reactions. CCR5-893(–) is a single-nucleotide deletion that results in complete truncation of the C tail of the gene product. We detected CCR5-893(–) in a sample of patients infected with non-tuberculosis mycobacteria and found that it was maintained heterozygously with a frequency of 2%. There is no association between this mutation and any immunodeficiency. Membrane expression of CCR5-893(–) was substantially reduced compared to the wild type, but this defective surface presentation recovered greatly recovered in the presence of 2 mg l-1 phytohemagglutinin (PHA). However, PHA inducement did not affect the total intracellular expression of CCR5-893(–) or wild-type CCR5. Thus we suggest there exist some PHA-induced factor(s) that could mediate the presentation of truncated CCR5.Received 23 July 2003; accepted 18 August 2003     

Defective phosphoinositide metabolism in primary hypertension

An increase in free cytosolic calcium content has been reported in essential hypertension. Since within the membrane, the phosphoinositides participate in the control of cell calcium homeostasis, we investigated whether impaired phosphoinositide metabolism could account for the calcium handling abnormality observed in hypertensives. In erythrocyte membranes of hypertensives the activity of kinases involved in polyphosphoinositide formation appears to be impaired and could be related to the alteration in calcium handling binding capacity and ATP-dependent calcium transport. In platelets of hypertensives, the hyperactivity of phospholipase C (observed even in the absence of calcium in the external medium) is likely to be responsible for the hypersensitivity of cells to various agonists. These observations are consistent with the hypothesis that in cells from hypertensives, a membrane defect linked to phosphoinositide metabolism is involved in the overall calcium handling defect.     

Nuclear localization of mouse fibroblast growth factor 2 requires N-terminal and C-terminal sequences

In vertebrates, different isoforms of fibroblast growth factor 2 (FGF2) exist, which differ by their N-terminal extension. They show different localization and expression levels and exert distinct biological effects. Nevertheless, genetic inactivation of all FGF2 isoforms in the mouse results in only mild phenotypes. Here, we analyzed mouse FGF2, and show that, as in the human, mouse FGF2 contains CTG-initiated high molecular-weight (HMW) isoforms, which contain a nuclear localization signal, and which mediate localization of this isoform to the nucleus. Using green fluorescent protein-FGF2 fusions, we furthermore observed, that C-terminal deletions disable nuclear localization of the short low-molecular-weight (LMW) 18-kDa isoform. This loss of specific localization is accompanied by a loss in heparin binding. We therefore suggest that, first, localization of mouse FGF2 is comparable to that in other vertebrates and, second, FGF2 contains at least two sequences important for nuclear localization, a nuclear localization sequence at the N terminus which is only contained in the HMW isoform, and another sequence at the C terminus, which is only required for localization of the LMW 18-kDa isoform. Received 1 July 2003; accepted 14 August 2003