Dual effects of guanosine 5′-[γ-thio]triphosphate on secretion by electroporated human neutrophils

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

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The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2771 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2771-2782 ◽

Cited By ~ 69

Author(s):

K A Joiner ◽

T Ganz ◽

J Albert ◽

D Rotrosen

Keyword(s):

Vitamin B12 ◽

Salmonella Typhimurium ◽

Human Serum ◽

Human Neutrophils ◽

Rabbit Igg ◽

Receptor Interactions ◽

Specific Granules ◽

Azurophil Granule ◽

Normal Human ◽

Specific Granule

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.

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Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42708-1 ◽

1992 ◽

Vol 267 (9) ◽

pp. 6396-6402

Author(s):

I Maridonneau-Parini ◽

J de Gunzburg

Keyword(s):

Plasma Membrane ◽

Cell Activation ◽

Human Neutrophils ◽

Specific Granules

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Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules

Biochemical Journal ◽

10.1042/bj2840513 ◽

1992 ◽

Vol 284 (2) ◽

pp. 513-520 ◽

Cited By ~ 7

Author(s):

S J Suchard ◽

M J Burton ◽

S J Stoehr

Keyword(s):

Cytochalasin B ◽

Anion Channel ◽

Polymorphonuclear Leucocyte ◽

Receptor Expression ◽

Human Neutrophils ◽

Ionophore A23187 ◽

Surface Receptors ◽

Specific Granules ◽

Azurophil Granule ◽

Disulphonic Acid

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

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R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE

10.1055/s-0038-1644504 ◽

1987 ◽

Author(s):

S K Joseph ◽

S Krishnamurthi ◽

V V Kakkar

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Kinase Inhibitor ◽

Human Platelets ◽

Maximal Concentration ◽

Inhibitory Effects ◽

Kinase C ◽

Granule Secretion ◽

Granule Release

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.

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Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes.

The Journal of Cell Biology ◽

10.1083/jcb.68.3.781 ◽

1976 ◽

Vol 68 (3) ◽

pp. 781-787 ◽

Cited By ~ 66

Author(s):

S Hoffstein ◽

R Soberman ◽

I Goldstein ◽

G Weissmann

Keyword(s):

Concanavalin A ◽

Binding Sites ◽

Polymorphonuclear Leukocytes ◽

Cytochalasin B ◽

Microtubule Assembly ◽

Human Neutrophils ◽

Con A ◽

Specific Granules ◽

Specific Granule ◽

Human Polymorphonuclear Leukocytes

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.

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ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES

The Journal of Cell Biology ◽

10.1083/jcb.28.2.277 ◽

1966 ◽

Vol 28 (2) ◽

pp. 277-301 ◽

Cited By ~ 281

Author(s):

Dorothy Ford Bainton ◽

Marilyn G. Farquhar

Keyword(s):

Bone Marrow ◽

Golgi Complex ◽

Polymorphonuclear Leukocyte ◽

Polymorphonuclear Leukocytes ◽

Dense Core ◽

Granule Formation ◽

Specific Granules ◽

Azurophil Granule ◽

Rabbit Bone ◽

Specific Granule

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO4. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (∼800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (∼500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.

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The Inhibitory Effect of Heparin and Related Glycosaminoglycans on Neutrophil Chemotaxis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661157 ◽

1984 ◽

Vol 52 (02) ◽

pp. 134-137 ◽

Cited By ~ 69

Author(s):

Yaacov Matzner ◽

Gerard Marx ◽

Ruth Drexler ◽

Amiram Eldor

Keyword(s):

Specific Binding ◽

Anticoagulant Activity ◽

Neutrophil Migration ◽

Inhibitory Effect ◽

Chemotactic Factor ◽

Human Neutrophils ◽

Boyden Chamber ◽

Neutrophil Chemotaxis ◽

Inhibitory Effects ◽

Chemotactic Factors

SummaryClinical observations have shown that heparin has antiinflammatory activities. The effect of heparin on neutrophil chemotaxis was evaluated in vitro in the Boyden Chamber. This method enabled differentiation between the direct effects of heparin on neutrophil migration and locomotion, and its effects on chemotactic factors. Heparin inhibited both the random migration and directed locomotion of human neutrophils toward zymosan-activated serum (ZAS) and F-met-leu-phe (FMLP). Inhibition was found to be dependent on the concentrations of the heparin and of the chemotactic factors. No specific binding of heparin to the neutrophils could be demonstrated, and heparin’s inhibitory effects were eliminated by simple washing of the cells. When added directly to the chamber containing chemotactic factor, heparin inhibited the chemotactic activity of ZAS but not that of FMLP, suggesting a direct inhibitory effect against C5a, the principal chemotactic factor in ZAS.Experiments performed with low-molecular-weight heparin, N-desulfated heparin, dextran sulfate, chondroitin sulfate and dextran indicated that the inhibitory effects of heparin on neutrophil chemotaxis are not related to its anticoagulant activity, but probably depend on the degree of sulfation of the heparin molecule.

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Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

Biochemical Journal ◽

10.1042/bj1820413 ◽

1979 ◽

Vol 182 (2) ◽

pp. 413-419 ◽

Cited By ~ 14

Author(s):

Holm Holmsen ◽

Linda Robkin ◽

H. James Day

Keyword(s):

Shape Change ◽

Adenine Nucleotides ◽

Human Platelets ◽

Dense Granule ◽

Metabolic Inhibitors ◽

Antimycin A ◽

Acid Hydrolase ◽

Acid Hydrolases ◽

Dense Granule Secretion ◽

Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

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