Abstract
Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.
Highlights
Polymorphonuclear leukocytes (PMN)1 are the predominant cell type present in areas of acute inflammation
Application of phagosomes to a ConA affinity column revealed that only a small fraction (Ͻ2–5%) of the vesicles was bound to the column, whereas intact PMN adhered to the affinity material (Ͼ95%), demonstrating that the vesicles had the inside-out orientation with little contamination of plasma membranes with exposed ConA receptors
Our findings on arachidonate export support the view [15, 28, 31] that transmembrane fatty acid movement is a protein-dependent and saturable process that cannot be explained by free diffusion
Summary
Polymorphonuclear leukocytes (PMN) are the predominant cell type present in areas of acute inflammation. The situation for PMN is complex since these cells do biosynthesize and release eicosanoids and avidly take up and metabolize arachidonic acid, LTA4 [1, 4], and LTB4 [12] To overcome those restrictions imposed by whole cell systems to the investigation of transport mechanisms, we prepared inside-out plasma membrane vesicles from PMN and investigated the usefulness of this system to study eicosanoid transport using arachidonic acid as a ligand. Five putative fatty acid transport proteins have been identified in the plasma membrane of several tissues including adipocytes, liver, endothelium, brain, intestine, kidney, lung, skeletal, and cardiac muscle For three of these cDNA clones have been isolated (reviewed in Ref. 15). With the transport assay presented here, it may be possible to separate the transport processes out of the complex cascade of events starting after activation of PMN by various stimuli to study the mechanism(s) of putative eicosanoid transporters and to investigate the impact of leukotriene inhibitors on transport
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