Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: Superoxide production without a rise in intracellular free calcium

1990 ◽  
Vol 145 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Songlin Liang ◽  
Timothy J. Woodlock ◽  
John C. Whitin ◽  
Marshall A. Lichtman ◽  
George B. Segel
Keyword(s):  
Signal Transduction ◽  
Concanavalin A ◽  
Superoxide Production ◽  
Intracellular Free Calcium ◽  
Human Neutrophils ◽  
Free Calcium

scholarly journals The dynamics of exocytosis in human neutrophils.

1988 ◽  
Vol 107 (6) ◽  
pp. 2117-2123 ◽  
Author(s):  
O Nüsse ◽  
M Lindau
Keyword(s):  
Calcium Concentration ◽  
Cytochalasin B ◽  
Intracellular Free Calcium ◽  
Morphological Data ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Membrane Area ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
Gamma S

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 microM guanosine-5'-O(3-thiotriphosphate) (GTP gamma S) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to integral of 8.4 pF corresponding to a 540 micron 2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTP gamma S-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and integral of 2.5 microM. In the absence of GTP gamma S elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP gamma S started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.

scholarly journals Defective signal-transduction pathways in T-cells from autoimmune MRL-lpr/lpr mice are associated with increased polyamine concentrations

1995 ◽  
Vol 311 (1) ◽  
pp. 175-182 ◽  
Author(s):  
T J Thomas ◽  
U B Gunnia ◽  
J R Seibold ◽  
T Thomas
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
Tyrosine Phosphorylation ◽  
Concanavalin A ◽  
Flow Cytometric Analysis ◽  
Signal Transduction Pathways ◽  
Free Calcium ◽  
Polyamine Biosynthesis ◽  
Beneficial Effects ◽  
Significant Difference

We previously reported that difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, exerted significant beneficial effects on the lifespan and disease expression of MRL-lpr/lpr mice, which spontaneously develop a lupus-like syndrome. Polyamine levels in splenic T-cells of MRL-lpr/lpr mice were significantly higher than those of Balb/c mice. In the present investigation, we examined the role of endogenous polyamines in transmembrane Ca2+ influx, generation of InsP3 and tyrosine phosphorylation of the p56lck protein in concanavalin A-stimulated splenic T-cells. Cytosolic free calcium concentrations ([Ca2+]i) in concanavalin A-stimulated T-cells of MRL-lpr/lpr and Balb/c mice were 250 +/- 25 and 450 +/- 42 nM respectively. Treatment of MRL-lpr/lpr mice with DFMO increased [Ca2+]i to 360 +/- 30 nM (P < 0.05). InsP3 levels of concanavalin A-stimulated MRL-lpr/lpr splenic T-cells were only 20% higher than those of unstimulated controls, whereas those of Balb/c T-cells were 90% higher. DFMO treatment increased InsP3 levels in concanavalin A-treated MRL-lpr/lpr T-cells to 67%. Western-blot analysis showed a 7-fold higher level of p56lck phosphorylation of MRL-lpr/lpr splenic T-cells than that of Balb/c mice. DFMO treatment reduced tyrosine phosphorylation of p56lck of MRL-lpr/lpr mice significantly (P < 0.001). Two-colour flow-cytometric analysis revealed no significant difference in the CD4+/CD8+ ratio in splenic T-cells of MRL-lpr/lpr mice after DFMO treatment. Polyamine levels in splenocytes were significantly reduced by DFMO treatment. These data show that DFMO treatment could alter signal-transduction pathways of splenic T-cells of MRL-lpr/lpr mice. Increased levels of polyamines in T-cells of untreated lpr mice contribute to defective signal-transduction pathways and the pathogenesis of lupus-like symptoms.

Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium

1996 ◽  
Vol 59 (6) ◽  
pp. 902-907 ◽  
Author(s):  
åsa Wilsson ◽  
Helen Lundqvist ◽  
Mikael Gustafsson ◽  
Olle Stendahl
Keyword(s):  
Staphylococcus Aureus ◽  
Intracellular Free Calcium ◽  
Human Neutrophils ◽  
Free Calcium

scholarly journals Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 522-531 ◽  
Author(s):  
GP L'Heureux ◽  
S Bourgoin ◽  
N Jean ◽  
SR McColl ◽  
PH Naccache
Keyword(s):  
Signal Transduction ◽  
Phospholipase D ◽  
Kinase Inhibitors ◽  
Interleukin 8 ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Cd11b Expression ◽  
Necrosis Factor Alpha ◽  
Herbimycin A ◽  
Transduction Mechanisms

Abstract Interleukin-8 (IL-8) and the structurally related cytokines neutrophil- activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists. IL-8, NAP-2, and GRO alpha stimulated similar increases in the level of cytoplasmic free calcium. They were also shown to stimulate qualitatively similar increases in the levels of protein tyrosine phosphorylation. In contrast, only IL-8 enhanced the formation of phosphatidylethanol (PEt), the product catalyzed by phospholipase D (PLD) in the presence of ethanol. The formation of PEt stimulated by IL-8 was inhibited by pertussis toxin and the tyrosine kinase inhibitors erbstatin and herbimycin A. The ability of IL-8 to stimulate the activity of PLD was additively enhanced, or primed, by cytochalasin B and by tumor necrosis factor alpha. Although all three chemokines increased the level of free cytoplasmic calcium to the same extent, IL-8 was significantly more potent than either NAP-2 or GRO alpha with respect to its ability to enhance CD11b expression and to stimulate chemotactic and oxidative responses. The differences between IL-8, NAP-2, and GRO alpha in their ability to stimulate PLD is likely to be related to their respective binding affinities for the two IL-8 receptors (IL-8R-A and IL-8R-B). These results suggest that the signalling pathways activated by IL-8R-A and IL-8R-B diverge at a step preceding the activation of PLD.

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