Thrombin Stimulates Activation of the Cerebral 5-Lipoxygenase Pathway during Blood-Brain Cell Contact

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Abstract
The purpose of this study was to identify the trigger mechanism activating the 5-lipoxygenase pathway during blood–brain cell contact and to estimate the contribution of blood and brain cells to the cysteinyl–leukotriene (LT) biosynthesis observed under these conditions. Incubation of dissociated rat brain cells in Krebs-Henseleit solution for up to 60 min did not stimulate any detectable cysteinyl–LT biosynthesis. Incubation of recalcified rat whole blood in vitro for up to 60 min led to release of only small amounts of cysteinyl–LT into the serum samples. However, coincubation of dissociated rat brain cells with physiologically recalcified autologous whole blood triggered a time-dependent release of large amounts of immunoreactive cysteinyl–LT into the serum samples. By reverse-phase HPLC, immunoreactive cysteinyl–LT was identified as a mixture of LTC4, LTD4, and LTE4. The extent of the 5-lipoxygenase stimulation depended on the amount of autologous blood coincubated with the dissociated brain cells. Activation of the 5-lipoxygenase pathway also occurred with coincubation of dissociated rat brain cells with recalcified autologous plasma. Stimulation of cysteinyl–LT biosynthesis during blood–brain cell contact remained unaffected by aprotinin, but concentration-dependent inhibition by the structurally and functionally unrelated thrombin inhibitors D-Phe-Pro-Arg-CH2Cl and recombinant hirudin was seen. Finally, when dissociated rat brain cells were incubated in Krebs-Henseleit solution in the presence of human α-thrombin, a concentration-dependent release of cysteinyl–LT into the buffer samples was observed. These data demonstrate that, in rats, during blood–brain cell contact, stimulation of the 5-lipoxygenase pathway in brain cells proceeds via α-thrombin as effector molecule.