The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-B activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.Lipopolysaccharide (LPS), a glycolipid constituent of the outer membrane of gram-negative bacteria, initiates signaling cascades in cells such as macrophages and endothelial cells, leading to the release of cytokines and other inflammatory mediators during sepsis. Excessive production of these mediators can cause septic shock and multiple organ failure (55).A decade ago, CD14, a 55-kDa glycoprotein and monocyte differentiation antigen, was identified as an important LPS recognition molecule (60). CD14 alone, however, is unable to transduce the intracellular LPS signal, since CD14 is only tethered to the cytoplasmic membrane by a glycosyl phosphatidylinositol anchor and lacks a membrane-spanning domain (17). Members of a family of proteins, the mammalian homologues of the Drosophila Toll protein, were found to act as transmembrane coreceptors to CD14 in the cellular response to LPS (34). These Toll-like receptors (TLR) contain ectodomains with leucine-rich repeats, and their intracellular motifs are highly homologous to intracellular signaling domains of interleukin-1 receptor type I (IL-1RI) and IL-1RI accessory protein (reviewed in reference 5). Following dimerization of the TLR, these domains attract the adapter protein MyD88, which in turn recruits the IL-1R-associated kinase. Following this association, IL-1R-associated kinase phosphorylates tumor necrosis factor receptor-associated factor 6, which in turn attracts two more protein tyrosine kinases, transforming growth factor beta-activated kinase 1 (TAK-1)...

We have designed a drug that protects the liver from apoptotic cell death by organ-selective pharmacological generation of the bioregulatory agent, nitric oxide (NO). The discovery strategy involved three steps: identifying a diazeniumdiolate ion (R2N[N(O)NO]-, where R2N = pyrrolidinyl) that spontaneously decomposes to NO with a very short half-life (3 s) at physiological pH; converting this ion to a series of potential prodrug derivatives by covalent attachment of protecting groups that we postulated might be rapidly removed by enzymes prevalent in the liver; and screening the prodrug candidates in vitro and in vivo to select a lead and to confirm the desired activity. Of five cell types examined, only cultured hepatocytes metabolized O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) to NO, triggering cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and protecting the hepatocytes from apoptotic cell death induced by treatment with tumor necrosis factor-alpha (TNF alpha) plus actinomycin D. In vivo, V-PYRRO/NO increased liver cGMP levels while minimally affecting systemic hemodynamics, protecting rats dosed with TNF alpha plus galactosamine from apoptosis and hepatotoxicity. The results illustrate the potential utility of diazeniumdiolates for targeting NO delivery in vivo and suggest a possible therapeutic strategy for hepatic disorders such as fulminant liver failure.

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