scholarly journals Staurosporine clamps cytosolic free Ca2+ concentrations of human neutrophils

1992 ◽  
Vol 283 (2) ◽  
pp. 499-505 ◽  
Author(s):  
K Wong ◽  
L Kwan-Yeung ◽  
J Turkson
Keyword(s):  
Binding Assay ◽  
Radioligand Binding ◽  
Human Neutrophils ◽  
Radioligand Binding Assay ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Combination Studies ◽  
Peptide Stimulation ◽  
Linear Manner ◽  
Stimulation Of

The present studies indicate that 50 nM-10 microM-staurosporine increased cytosolic free Ca2+ concentrations ([Ca2+]i) of fura-2-loaded neutrophils in a non-linear manner. The rise in [Ca2+]i was rapid, reaching a plateau (e.g. to 0.4 microM with 1 microM-staurosporine) within 30 s, and was maintained for more than 20 min. Pretreating cells with pertussis toxin had no effect on this reaction. The elevation of [Ca2+]i was insensitive to extracellular Ca2+ concentrations and was due entirely to mobilization of intracellular Ca2+ stores. Mn(2+)-quench studies confirmed the absence of Ca2+ influx. No Ca2+ efflux occurred in staurosporine-treated cells. In combination studies, staurosporine potentiated Ca2+ influx induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) and did not block Ca2+ efflux associated with peptide stimulation of neutrophils. Studies with permeabilized cells showed that staurosporine did not directly release intracellular Ca2+ stores, nor did it affect the sequestration of Ca2+ by a Ca2+/ATPase pump. A radioligand-binding assay failed to detect changes in the level of inositol 1,4,5-trisphosphate in neutrophils incubated with less than or equal to 1 microM-staurosporine, but in cells treated with 10 microM-staurosporine the assay recorded a transient increase in this second messenger similar to that induced by FMLP. Finally, lysozyme, but not beta-glucuronidase, was released from staurosporine-treated cells. The present results suggest that staurosporine increased [Ca2+]i by indirectly mobilizing internal Ca2+ stores. Staurosporine suppression of Ca2+ efflux and generation of a persistent signal may account for the maintained elevation of [Ca2+]i.

scholarly journals GMP-140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction.

1991 ◽  
Vol 112 (3) ◽  
pp. 491-499 ◽  
Author(s):  
K L Moore ◽  
A Varki ◽  
R P McEver
Keyword(s):  
Binding Assay ◽  
Radioligand Binding ◽  
Leukocyte Adhesion ◽  
Specific Binding ◽  
Sialic Acids ◽  
Human Neutrophils ◽  
Radioligand Binding Assay ◽  
Surface Molecules ◽  
Activated Platelets ◽  
Selectin Family

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism.

A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin

2003 ◽  
Vol 279 (1-2) ◽  
pp. 173-181 ◽  
Author(s):  
Silvina N. Valdez ◽  
Rubén F. Iacono ◽  
Anabel Villalba ◽  
Alejandro Cardoso Landaburu ◽  
Mario R. Ermácora ◽  
...  
Keyword(s):  
Binding Assay ◽  
Radioligand Binding ◽  
Radioligand Binding Assay

scholarly journals The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil

1990 ◽  
Vol 271 (1) ◽  
pp. 209-213 ◽  
Author(s):  
N T Thompson ◽  
J E Tateson ◽  
R W Randall ◽  
G D Spacey ◽  
R W Bonser ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Human Neutrophil ◽  
Second Messengers ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Inositol 1,4,5 Trisphosphate ◽  
Phosphatidate Phosphohydrolase ◽  
Time Courses ◽  
Rapid Activation ◽  
Stimulation Of

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.

scholarly journals A radioligand binding assay for the insulin-like growth factor 2 receptor

PLoS ONE ◽  
2020 ◽  
Vol 15 (9) ◽  
pp. e0238393
Author(s):  
Pavlo Potalitsyn ◽  
Irena Selicharová ◽  
Kryštof Sršeň ◽  
Jelena Radosavljević ◽  
Aleš Marek ◽  
...  
Keyword(s):  
Growth Factor ◽  
Binding Assay ◽  
Radioligand Binding ◽  
Insulin Like Growth Factor ◽  
Radioligand Binding Assay
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