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Binding and metabolism of leukotriene B 4 by neutrophils and their subcellular organelles
Author(s) -
O'Flaherty Joseph,
Kosfeld Scott,
Nishihira Jun
Publication year - 1986
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041260306
Subject(s) - degranulation , percoll , cytoplasm , chemistry , biochemistry , differential centrifugation , endoplasmic reticulum , extracellular , leukotriene , cell fractionation , membrane , centrifugation , biology , receptor , immunology , asthma
The subcellular distribution of leukotriene (LT)B 4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [ 3 H]LTB 4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB 4 analogues at concentrations corresponding to their respective potencies in (1) blocking [ 3 H]LTB 4 binding to whole cells and (2) stimulating neutrophil degranulation responses. In contrast, [ 3 H]LTB 4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4°C. The cell‐free system, like intact cells, metabolized [ 3 H]LTB 4 to ω‐oxidized product rapidly and quantitatively at 37°C but was inactive at 4°C. Whole cells converted radiolabel to 20‐hydroxy (∼ 30% of product) and 20‐carboxy (∼ 70% of product) derivatives; the cell‐free system formed principally 20‐hydroxy‐[ 3 H]LTB 4 . These products were less bioactive than LTB 4 . Nevertheless, metabolism of LTB 4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB 4 within 3–5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB 4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate ω‐oxidase. At the level of the individual cell, receptor down‐regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.