Hepoxilin A₃-specific binding in human neutrophils Available to Purchase

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 K_D and B_max. 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.