THROMBIN STIMULATES ACTIVATION OF THE CEREBRAL 5-LIPOXYGENASE PATHWAYDURING BLOOD-BRAIN CELL CONTACT

The purpose of this study was to identify the trigger mechanism activa ting the 5-lipoxygenase pathway during blood-brain cell contact and to estimate the contribution of blood and brain cells to the cysteinyl-l eukotriene (LT) biosynthesis observed under these conditions. Incubati on of dissociated rat brain cells in Krebs-Henseleit solution for up t o 60 min did not stimulate any detectable cysteinyl-LT biosynthesis. I ncubation 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 sampl es. However, coincubation of dissociated rat brain cells with physiolo gically 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 ident ified as a mixture of LTC(4), LTD(4), and LTE(4). The extent of the 5- lipoxygenase stimulation depended on the amount of autologous blood co incubated with the dissociated brain cells. Activation of the 5-lipoxy genase pathway also occurred with coincubation of dissociated rat brai n cells with recalcified autologous plasma. Stimulation of cysteinyl-L T 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 cel ls were incubated in Krebs-Henseleit solution in the presence of human alpha-thrombin, a concentration-dependent release of cysteinyl-LT int o the buffer samples was observed. These data demonstrate that, in rat s, during blood-brain cell contact, stimulation of the 5-lipoxygenase pathway in brain cells proceeds via alpha-thrombin as effector molecul e.