Superoxide release by Zymosan-stimulated Rat Kupffer cells in vitro

Abstract

Kupffer cells were isolated from pronase-perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O₂ uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol-dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophophate shunt were observed. Chemiluminescence and O₂ production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan-induced chmiluminesceance was not inhibited in the presence of the non-penetrating thiol reagents, 5,5′-dithio-bis-2-nitrobenzoate and iodoacetyl-sepharose. Iodoacetate acted on the cytosolic glucose-6-phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.