Hepatic cytokine gene expression is independently stimulated by circulating microbial products and reductions in the cellular O2 supply. Although these stimuli occur sequentially after gram-negative bacteremia, it is unknown whether their interplay augments production of interleukin (IL)-1 by the liver. We studied the effects of intraportal Escherichia coli (EC) bacteremia and secondary constant-flow hypoxia (Po2, approximately 46 Torr for 30 min) on IL-1 alpha and IL-1 beta gene expression in ex situ buffer-perfused rat livers over 180 min (n = 67). At t = 0, normoxic EC and normal saline (NS) controls received 10(9) live EC serotype 055:B5 and 0.9% NaCl, respectively; in livers subjected to EC+hypoxia-reoxygenation (H/R) and NS+H/R, hypoxia began 0.5 h after EC or NS and was followed by 120 min of reoxygenation. Portal and hepatic venous perfusates were serially analyzed for bacterial colony-forming units, O2 uptake, and aspartate aminotransferase. At 60 min (peak hypoxia) and 180 min, cDNAs for IL-1 alpha and IL-1 beta were hybridized to whole liver RNA, and IL-1 beta protein levels in venous perfusates were assessed. Intrahepatic levels of reduced glutathione (GSH) were measured as an index of oxidative stress. Compared with normoxic EC, IL-1 alpha transcripts decreased at 180 min in EC+H/R livers (P < 0.0001) as did IL-1 beta mRNA (P < 0.05), despite similar EC clearance, GSH levels, posthypoxic O2 uptake, and aspartate aminotransferase release. Hepatic secretion of IL-1 beta likewise fell in EC+H/R vs. EC controls (P < 0.005). Prostaglandin H synthase-2 (COX-2) message accumulation was not enhanced by H/R, and indomethacin did not reverse H/R-mediated suppression of IL-1 production. In contrast, H/R-related falls in EC-induced IL-1S expression were reversed by allopurinol or catalase. Thus brief hypoxic stress of the liver causing neither GSH depletion nor functional impairment downregulates postbacteremic IL-1 expression by a mechanism involving O2 radicals but not cyclooxygenase metabolites.

Activity in trypsin-like proteases is the result of proteolytic cleavage at R15 followed by an ionic interaction that ensues between the new N terminus of I16 and the side chain of the highly conserved D194. This mechanism of activation, first proposed by Huber and Bode, organizes the oxyanion hole and primary specificity pocket for substrate binding and catalysis. Using the clotting protease thrombin as a relevant model, we unravel contributions of the I16-D194 ionic interaction to Na+ binding, stability of the transition state and the allosteric E*-E equilibrium of the trypsin fold. The I16T mutation abolishes the I16-D194 interaction and compromises the architecture of the oxyanion hole. The D194A mutation also abrogates the I16-D194 interaction but, surprisingly, has no effect on the architecture of the oxyanion hole that remains intact through a new H-bond established between G43 and G193. In both mutants, loss of the I16-D194 ionic interaction compromises Na+ binding, reduces stability of the transition state, collapses the 215–217 segment into the primary specific pocket and abrogates the allosteric E*-E equilibrium in favor of a rigid conformation that binds ligand at the active site according to a simple lock-and-key mechanism. These findings refine the structural role of the I16-D194 ionic interaction in the Huber-Bode mechanism of activation and reveal a functional linkage with the allosteric properties of the trypsin fold like Na+ binding and the E*-E equilibrium.

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