Thrombin stimulates activation of the cerebral 5-lipoxygenase pathway during blood-brain cell contact

The purpose of this study was to identity 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 alpha-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 alpha-thrombin as effector molecule.