Thrombocytes are the predominant source of endogenous sulfidopeptide leukotrienes in the American bullfrog (Rana catesbeiana).

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.

Endogenous sulfidopeptide leukotriene synthesis and 12-lipoxygenase activity in bullfrog (Rana catesbeiana) erythrocytes.

Endogenous leukotriene (LT) synthesis by mammalian inflammatory cells requires both 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein. Other myeloid cells, like erythrocytes, have an incomplete 5-lipoxygenase pathway and synthesize leukotrienes transcellularly. Several studies indicate that sulfidopeptide leukotrienes have important physiological functions in bullfrogs and receptors have been characterized. Calcium ionophore activated bullfrog blood was analyzed by reverse phase-high-performance liquid chromatography (RP-HPLC). Endogenous metabolites consisted of 5-LO products including leukotriene D4. Other metabolites also suggested 12-lipoxygenase activity. Following purification, metabolites from activated erythrocytes were analyzed by RP-HPLC coupled with radioimmunoassay. Erythrocytes demonstrated endogenous synthesis of LTD4 which was inhibited by non-selective (NDGA) and specific (MK886) 5-lipoxygenase inhibitors. Experiments with partially purified erythrocyte cytosol further confirmed 5-LO activity and revealed 12-lipoxygenase activity. HPLC analysis of [1-14C]arachidonic acid labeled metabolites from activated erythrocytes indicates that most of the available substrate is converted to 12-hydroxy-eicosatetraenoic acid (12-HETE). These novel findings indicate that, in contrast to mammals, bullfrog erythrocytes endogenously synthesize LTD4 and large quantities of 12-HETE giving them the potential to contribute directly to inflammatory responses. The evolutionary loss of the nucleus in mammalian erythrocytes appears to be associated with the inability to synthesize leukotrienes endogenously.