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

Abstract

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.

Cysteinyl-Leukotriene Levels in Intracerebral Hemorrhage:An Edema-Promoting Factor?

“…This is in analogy with results obtained with human astrocytoma tissue slices which predominantly released LTD 4 and LTE 4 and smaller amounts of LTC 4 [30]. In dissociated rat brain cells mainly LTC 4 and smaller amounts of LTD 4 have been detected [37]. The reason for the difference in HPLC profiles between cys-LT excreted in patients’ urine and rat brain tissue is the advanced degradation of the highly potent cys-LT from LTC 4 to the metabolites LTD 4 and LTE 4 .…”

“…In patients suffering from vasospasm after aneurysmal subarachnoid hemorrhage significantly higher cisternal cerebrospinal fluid levels of cys-LT were observed as compared to patients without vasospasm suggesting a participation of cys-LT under these pathophysiological conditions [21, 39]. The coincubation of physiologically recalcified whole blood with autologous dissociated rat brain cells triggers a time- and volume-dependent biosynthesis of cys-LT [37]. In contrast, incubation of rat brain cells without an external stimulus is not accompanied by any detectable release of cys-LT [36].…”

“…Their release provokes increased vascular permeability or stimulation of leukocyte adhesion [6]. We have recently shown that coincubation of blood and dissociated brain cells may lead to a time-dependent and enhanced formation of cys-LT in rats [37]. We could further show that the activation of the 5-lipoxygenase pathway proceeds via alpha thrombin as the effector molecule [26, 27].…”

Effects of an Antithrombin Drug in Patients with Subacute Exacerbations of Binswanger Disease.

“…Microcirculation distubance is caused by thrombin generation in the brain. In addition, thrombin may directly damage nervous tissues by an increase of vascular permeability (23) and nitric oxide production in glial cells, induction of apoptosis in neurons (24) and suppression of neurite outgrowth (25). Argatroban treatment increases cerebral blood flow in ischemic brains (26,27) and is also expected to ameriolate these direct effects of thrombin.…”

Thrombin and Antithrombin in Binswanger''s Disease

“…Nishino et al reported that thrombin infusion into the rat caudate nucleus causes increased reactive gliosis, infiltration of inflammatory cells, proliferation of mesenchymal cells, and induction of angiogenesis (8). In addition, thrombin may directly promote tissue damage in the central nervous system through an increase of vascular permeability and nitric oxide synthesis in glial cells (9). Donovanet al have shownthat thrombincauses apoptosis as evidenced by cleavage of DNA,fragmentation of nuclei, and prevention of death by inhibition of protein synthesis (10).…”