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 prot ein. Other myeloid cells, like erythrocytes, have an incomplete 5-lipo xygenase pathway and synthesize leukotrienes transcellularly. Several studies indicate that sulfidopeptide leukotrienes have important physi ological functions in bullfrogs and receptors have been characterized. Calcium ionophore activated bullfrog blood was analyzed by reverse ph ase-high-performance liquid chromatography (RP-HPLC). Endogenous metab olites consisted of 5-LO products including leukotriene D-4. Other met abolites also suggested 12-lipoxygenase activity. Following purificati on, metabolites from activated erythrocytes were analyzed by RP-HPLC c oupled with radioimmunoassay. Erythrocytes demonstrated endogenous syn thesis of LTD, which was inhibited by non-selective (NDGA) and specifi c (MK886) 5-lipoxygenase inhibitors. Experiments with partially purifi ed erythrocyte cytosol further confirmed 5-LO activity and revealed 12 -lipoxygenase activity. HPLC analysis of [1-C-14]arachidonic acid labe led 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 LTD, and large quantiti es of 12-HETE giving them the potential to contribute directly to infl ammatory responses. The evolutionary loss of the nucleus in mammalian erythrocytes appears to be associated with the inability to synthesize leukotrienes endogenously.