Verotoxin and ricin have novel effects on preproendothelin-1 expression but fail to modify nitric oxide synthase (ecNOS) expression and NO production in vascular endothelium

Interaction of bipartite Escherichia coli O157-derived verotoxins (VTs) 1 and 2 (Shiga toxin 1 and 2) with vascular endothelium is believed to play a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome and of E. coli O157-associated hemorrhagic colitis. We defined the effects of VTs on the expression of potent endothelial cell-derived regulators of vascular wall function, namely endothelin-l (ET-1) and nitric oxide (NO). In quiescent bovine aortic endothelial cells, both VT1 and VT2, but not receptor-binding VT B-subunit which lacks N-glycosidase activity, induced concentration-dependent (0.1-10 nM) increases in steady state preproET-1 mRNA transcript levels, an effect that was maximal at 12-24 h. Metabolic-labeling experiments indicated that VTs increased preproET-1 mRNA transcript levels at concentrations that had trivial effects on nascent DNA, RNA, and protein synthesis. In contrast to preproET-1, endothelin converting enzyme-1 and endothelial constitutive NO synthase mRNA transcript levels remained unchanged. Consistent with these findings, VTs failed to modulate immunoreactive endothelial constitutive NO synthase expression and basal and calcium-dependent L-[C-14]arginine to L-[C-14]citrulline conversion or the NO chemiluminescence signal. The plant-derived toxin ricin, which shows a similar molecular mechanism of enzymatic ribosomal modification to VTs, caused comparable effects on these endothelial vasomediators and metabolite incorporation, at 3 log orders lower concentrations. Nuclear transcription and actinomycin D chase experiments indicated that VTs stabilize labile preproET-1 mRNA transcripts in endothelial cells, Therefore, VTs potently increase select mRNA transcript levels in endothelial cells at concentrations of toxins that have minimal effects on protein synthesis. Perturbed expression of endothelial-derived vasomediators may play a pathophysiologic role in the microvascular dysfunction that is the hallmark of hemolytic uremic syndrome and hemorrhagic colitis.