Nucleotide-, chemotactic peptide- and phorbol ester-induced exocytosis in HL-60 leukemic cells

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5% of the G-proteins. Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca2+ and beta-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma S]-induced beta-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma S], UTP and PMA did not induce beta-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma S] induced a transient shape change. Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB. (II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS

The transduction processes of human neutrophil superoxide generation activated by receptor and post-receptor mechanisms

The generation of toxic oxygen radicals, by activated human neutrophils, is involved not only in microbial killing but has been implicated in tissue injury. The development of improved therapeutic agents necessitates an adequate understanding of the events coupling receptor stimulation with oxygen radical production. At least two signal-transduction pathways appear to be involved in the generation of oxygen radicals. One involves the phospholipase C mediated breakdown of phosphatidylinositol bisphosphate - giving rise to inositol trisphosphate (which mobilizes calcium) and diacylglycerol (which activates protein kinase C). The other pathway involves generation of diradylglycerols from non-inositol containing lipids by the action of phospholipase D. However derived, increased diradylglycerol levels and subsequent protein kinase C activation have been implicated in the generation of superoxide (O2-). Despite this, a controversy still exists as to the involvement of protein kinase C in receptor-mediated O2- production. Superoxide was measured by the reduction of ferricytochrome c initiated by receptor stimuli (fMLP, opsonized zymosan, IgG and heat-aggregated IgG), two direct protein kinase C activators (OAG and DiC8), a calcium ionophore (A23187) and a compound thought to mobilize membrane lipids (y-HCCH). Newly available and reportedly specific protein kinase C inhibitors, and putative inhibitors of diacylglycerol metabolism were used to investigate the transduction mechanisms. The results suggest that contrary to reports in the literature, protein kinase C has a central role in the signal transduction of the respiratory burst. Differences in the transduction pathways for the receptor stimuli were found and an arachidonate pathway may play a role in opsonized zymosan- and IgG-induced O2- generation. This raises the possibility that drugs could be designed which might reduce toxic oxygen radical-mediated tissue damage (such as that caused by immune complexes), without reducing the effectiveness of microbial killing. The ability of the non-steroidal anti-inflammatory drugs (NS AIDs) to modify O2- production was also investigated. Some NSAIDs, namely sodium meclofenamate, mefanamic acid and benoxaprofen, actually exacerbated superoxide release when neutrophils were stimulated by both receptor and post-receptor mechanisms. Indomethacin potentiated post-receptor stimulated O2- generation, but inhibited fMLP, Fe and C3b receptor-mediated O2- production. These results could have clinical relevance

Purinergic signaling in the modulation of redox biology

Purinergic signaling is a cell communication pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides are released in physiological and pathological circumstances activating purinergic type 2 receptors (P2 receptors): P2X ion channels and P2Y G protein-coupled receptors. The activation of these receptors triggers the production of reactive oxygen and nitrogen species and alters antioxidant defenses, modulating the redox biology of cells. The activation of P2 receptors is controlled by ecto-enzymes named ectonucleotidases, E-NTPDase1/CD39 and ecto-5’-nucleotidase/CD73) being the most relevant. The first enzyme hydrolyzes adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP), and the second catalyzes the hydrolysis of AMP to adenosine. The activity of these enzymes is diminished by oxidative stress. Adenosine actives P1 G-coupled receptors that, in general, promote the maintenance of redox hemostasis by decreasing reactive oxygen species (ROS) production and increase antioxidant enzymes. Intracellular purine metabolism can also contribute to ROS generation via xanthine oxidase activity, which converts hypoxanthine into xanthine, and finally, uric acid. In this review, we describe the mechanisms of redox biology modulated by purinergic signaling and how this signaling may be affected by disturbances in the redox homeostasis of cells

Identification and characterization of Haemophilus somnus components which interfere with bovine polymorphonuclear leukocyte functions

The components present in Haemophilus somnus fractions which suppressed Staphylococcus aureus ingestion and/or iodination of protein by bovine polymorphonuclear leukocytes (PMNs) were identified and characterized. Two fractions which suppressed iodination but not ingestion were isolated by heat-extracting a washed bacterial suspension at 60 C or incubating the bacterial suspension at 37 C and were partially purified by ultrafiltration (HElF-60 and HElF-37 respectively). The components present in these fractions were separated by reverse-phase high performance liquid chromatography (HPLC) and identified as ribonucleotides, a ribonucleoside, and purine and pyrimidine bases. It was found that guanine and guanosine 5\u27-monophosphate (GMP) were the components responsible for most of the suppression in HElF-60 while guanine and adenine were the major inhibitory components in HElF-37;Through purification by HPLC (anion exchange and reverse-phase) and characterization by chemical analyses, the active component present in the fraction which suppressed both iodination and ingestion by PMNs was identified as ribonucleic acid (RNA). A significant quantity of antibodies were removed from antiserum which contained antibodies to RNA by absorption with Formalin-killed H. somnus. This suggested that RNA was present on the surface of H. somnus cells. Other RNA preparations of different molecular species or sources were also suppressive to both iodination and ingestion;In order to determine if cattle can be induced to produce antibodies against guanine, adenine, and GMP, groups of Holstein calves were immunized with GMP-keyhold limpet hemocyanin conjugate (GMP-KLH), guanosine-KLH, or adenosine-KLH. High titers of antibody to GMP, guanosine, or adenosine developed following the administration of two doses of each antigen. Anti-guanosine antibody crossreacted with deoxyguanosine, guanine, GMP and yeast RNA but not with adenosine or adenine. Similarly, anti-adenosine antibody was found to crossreact with deoxyadenosine, adenine, adenosine 5\u27-monophosphate, and yeast RNA but not with guanosine or guanine. In contrast, anti-GMP antibody crossreacted only with deoxy-GMP

Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acidâ stimulated neutrophils

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142119/1/jlb0976.pd

The biochemical role of the small G protein Rac1 in cell signalling pathways - Interaction with RhoGDI and the phagocyte NADPH oxidase component, p67phox

Rac is a small G protein with a number of signalling roles. Along with other members of the Rho family of small GTPases, it is involved in the control of the actin cytoskeleton (Hall, 1992) and an apparently separate role in the activation of the NADPH oxidase, an enzymatic mechanism in phagocytes which forms superoxide in response to bacterial infection. In an inactive state, Rho family proteins exist in a complex with a second cytosolic protein, Rho guanine nucleotide dissociation inhibitor, RhoGDI. Activation causes dissociation of the Rac GDI complex and movement of Rac to the membrane. Spectroscopic studies have been used to investigate the interaction of Racl with other molecules, such as p67phox, a component of the NADPH oxidase complex. Complexes of racl with 2'(3')O-(N-methylanthraniloyl) (mant) fluorescent nucleotide analogues (eg. mantGDP) have been used to try to develop methods to study the interaction of Racl and p67phox. Although a previous report indicates a fluorescent change when Racl (complexed to a fluorescent nucleotide analogue) is incubated with p67phox, these experiments could not be repeated. A number of other approaches have been taken to develop a system to monitor the interaction of Rac and p67phox. Fluorescent approaches have also been developed to study the interaction of Rac and RhoGDI. GDI has previously been labelled with the fluorophore N-[2-1-(maleimidyl)ethyl]-7- (diethylamino)coumarin-3-carboxamide (MDCC) on a single cysteine in our laboratory and shows a large fluorescence decrease on Rac binding. Rac requires a lipid modification at the C-terminus to interact with RhoGDI, which presents a number of experimental difficulties. A system has been developed using C-terminally truncated (E.coli expressed) Rac and a farnesylated C-terminal peptide that mimics full length Racl that has been lipid modified in vivo (Newcombe et al., 1999). We are currently using this system to study the interaction of Racl with GDI and a number Racl point mutants have been made in the major regions of divergence between Ras superfamily proteins, based on the crystal structure of Racl (Hirshberg et al., 1997). In addition, a Racl/H-Ras chimaeric protein has been made and expressed in this laboratory. Results indicate that a region of the Racl effector loop is important for the Racl GDI interaction, with mutations in the insertion loop of Racl having little or no affect on the affinity of the Racl GDI interaction (Newcombe, Hunter & Webb, unpublished results). In addition, the interaction of Racl with a number of novel fluorescent nucleotide analogues including 3'-O-[N-[2-(7-diethylaminocoumarin-3-carboxamido)ethyl]carbamoyl] GTP (cou-edaGTP) and coumarin343-edaGTP (but-edaGTP) have been tested, and Racl complexes have shown that the rate of nucleotide hydrolysis and exchange by Racl shows them to be good analogues of GTP. It is hoped that these analogues will be useful to study the interaction of Racl with other proteins, such as GDI and p67phox

Cyclic AMP Inhibits Secretion From Electroporated Human Neutrophils

It has long been known that Intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re‐examine the effects of cyclic nucleotides on Ca2+‐induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor‐ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 μM inhibited Ca2+‐induced secretion; 30–300 μM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane‐permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 μM and modestly inhibitory at 300 μM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half‐maximal inhibition by cAMP occurred at 10–30 μM. Inhibition by cAMP was achieved by shifting the Ca2+ dose‐response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B‐glucuronidase). We previously showed that ATP could enhance Ca2+‐induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141578/1/jlb0172.pd

Papel del ácido oleico en el sistema inmune: mecanismos de acción: revisión científica

Introduction:Although n-3 polyunsaturated fatty acids have been widely described as antiinflammatory fats, little is known about the role of oleic acid in immune system. Aim: The aim of the present review is to join all the reports available in order to analyze where exactly the knowledge concerning this topic is and what the causes of the controversial data could be. Methods: We searched electronic databases and bibliographies of selected articles were inspected for further reference. Results:Diets rich in oleic acid have beneficial effects in inflammatory-related diseases. In addition, a wide range of studies evaluate the effect of oleic acid in different cellular functions thus reporting a potential mechanism for the biological effect of such a fat. However, some controversial data can be found in literature, maybe related to the kind of study or even the dose of the reagent added. Conclusion:In conclusion, oleic acid could be reported as an anti-inflammatory fatty acid playing a role in the activation of different pathways of immune competent cells