In this study we aimed to investigate whether the therapeutic efficacy of
anisodamine in the treatment of bacteraemic
shock could--at least in part--be brought about by its direct interference with the
lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of
anisodamine on LPS-induced expression of
plasminogen activator inhibitor-1 (PAI-1) and
tissue factor (TF), two major markers of endothelial activation.
PAI-1 was measured in the
conditioned media of human umbilical vein endothelial cells (HUVEC) by a specific
enzyme-linked
immunosorbent assay (ELISA) whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Results obtained in these assays were confirmed on the level of specific
mRNA expression by Northern blotting using specific probes for human
PAI-1 or TF. In order to evaluate a possible contribution of the
NF-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and
NF-kappa B-binding
oligonucleotides. When HUVEC were treated with 1 microg/ml LPS a significant increase in
PAI-1 and TF activity was observed compared with cells incubated without LPS.
Anisodamine dose-dependently inhibited this LPS-induced upregulation of
PAI-1 and TF.
Anisodamine alone had no effect on the constitutive expression of
PAI-1 and TF in these cells. These effects were also confirmed on the level of specific
PAI-1 and TF
mRNA expression by Northern blotting. Furthermore, we could show by EMSA that
anisodamine completely abolished LPS-induced
NF-kappa B DNA binding activity in nuclear extracts from HUVEC treated with LPS together with
anisodamine. Thus, we provide evidence that
anisodamine counteracts endothelial cell activation by inhibiting LPS-induced
PAI-1 and TF expression in these cells. Its interference with the
NF-kappa B pathway might - at least in part - contribute to this effect. The ability of
anisodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic
shock.