Comparative effect of leukotriene B4 and leukotriene B5 on calcium mobilization in human neutrophils

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Download Full-text

Calcium mobilization in fluoride activated human neutrophils

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(85)91855-8 ◽

1985 ◽

Vol 133 (1) ◽

pp. 161-167 ◽

Cited By ~ 46

Author(s):

Colette F. Stranad ◽

Kenneth Wong

Keyword(s):

Calcium Mobilization ◽

Human Neutrophils

Download Full-text

Phospholipase A2 activation in human neutrophils requires influx of extracellular Ca2+ and leukotriene B4

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.1.c138 ◽

1995 ◽

Vol 268 (1) ◽

pp. C138-C146 ◽

Cited By ~ 23

Author(s):

S. Reddy ◽

R. Bose ◽

G. H. Rao ◽

M. Murthy

Keyword(s):

Ethylene Glycol ◽

Phospholipase A2 ◽

Platelet Activating Factor ◽

Leukotriene B4 ◽

Lipid Mediators ◽

Oxidation Products ◽

Human Neutrophils ◽

Tetraacetic Acid ◽

A 23187 ◽

Transient Elevation

We have demonstrated that phospolipase A2 (PLA2) activation in human neutrophils requires both the influx of extracellular Ca2+ and leukotriene B4 (LTB4). Surprisingly, the eicosanoids (LTB4 and its omega-oxidation products) formed were quantitatively very similar in both thapsigargin (Thap)- and A-23187-stimulated neutrophils. In contrast, Thap had very little effect on the activation of PLA2 when 5-lipoxygenase (5-LO) was blocked by BW755C or MK-886, whereas A-23187 caused a substantial activation. The lack of PLA2 activation in Thap-stimulated neutrophils results from the inhibition of LTB4 formation in the presence of 5-LO inhibitors. It appears that A-23187 activates both LTB4-dependent and -independent PLA2, whereas Thap activates LTB4-dependent PLA2. Experiments with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid demonstrated that activation of Thap-sensitive PLA2 and 5-LO requires the influx of Ca2+. Neither the transient elevation of cytosolic Ca2+ from intracellular stores nor the sustained Ca2+ influx alone without LTB4 appears sufficient to cause the activation of LTB4-dependent PLA2. We suggest that the activation of LTB4-dependent PLA2 involves 1) a sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+ and 2) a coupling between LTB4 and its receptor. We conclude that LTB4-dependent PLA2 plays an important role in the poststimulatory formation of lipid mediators such as prostaglandins, leukotrienes, and platelet-activating factor.

Download Full-text