Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro-inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll-like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron-glia cultures with antibodies against F4/80 revealed that while TLR4(+/+) microglia were activated the low concentration of 1 ng/ml of LPS, TLR4(-/-) microglia exhibit activated morphology in response to LPS only at higher concentrations (100-1,000 ng/ml). Additionally, tumor necrosis factor-alpha (TNF-alpha) was only produced from higher concentrations (100-1,000 ng/ml) of LPS in TLR4(-/-) enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF-alpha production from TLR4(-/-) microglia. The influence of TLR4 on LPS-induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4(-/-) and TLR4(+/+) microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1-1,000 ng/ml). These data indicate that LPS-induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-alpha response of both the TLR4-dependent and independent pathway.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.Cellular inositol phosphate metabolism is an intricate web of kinase and phosphatase reactions that produces a number of important signaling molecules (for review see Ref. 1). A now classic example of these signaling activities is the release of Ca 2ϩ into the cytoplasm through an intracellular channel that is gated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 4 (2). Additional roles are continually being discovered: inositol phosphates have recently been shown to be critical to the activity of RNA-editing enzymes (3), to participate in telomere maintenance (4), and to be the phosphate donors in certain protein phosphorylation events (5). It is of critical interest, therefore, to establish the regulatory mechanisms that govern the metabolism of inositol phosphates.An interesting feature of inositol phosphate metabolism is the promiscuity with which several key kinases phosphorylate multiple substrates (6). For example, ITPK1 (also known as inositol 1,3,4-trisphosphate 5/6-kinase) adds either a 5-or 6-phosphate to Ins(1,3,4)P 3 and also attaches a 1-phosphate to Ins(3,4,5,6)P 4 (6, 7) (inositol phosphate structures shown in Fig. 2). These reactions have been demonstrated to be reversible: ITPK1 can also dephosphorylate Ins(1,3,4,5,6)P 5 back to Ins(3,4,5,6)P 4 (8). An especially puzzling aspect of this phenomenon is that dephosphorylation of Ins(1,3,4,5,6)P 5 by human ITPK1 is stimulated, rather than competitively inhibited, by one of its alternate substrates, Ins(1,3,4)P 3 (8).The fact that mammalian ITPK1 reversibly phosphorylates both Ins(3,4,5,6)P 4 and Ins(1,3,4)P 3 takes on...

Orally delivered salt stimulates renal salt excretion more effectively than does iv delivered salt. Although the mechanisms that underlie this "postprandial natriuresis" are poorly understood, the peptide uroguanylin (UGn) is thought to be a key mediator. However, the lack of selective assays for UGn gene products has hindered rigorous testing of this hypothesis. Using peptide-specific assays, we now report surprisingly little UGn in rat intestine or plasma. In contrast, prouroguanylin (proUGn), the presumed-inactive precursor of UGn, is plentiful (at least 40 times more abundant than UGn) in both intestine and plasma. The intestine is the likely source of the circulating proUGn because: 1) the proUGn portal to systemic ratio is approximately two under normal conditions, and 2) systemic proUGn levels decrease rapidly after intestinal resection. Together, these data suggest that proUGn itself is actively involved in enterorenal signaling. This is strongly supported by our observation that iv infusion of proUGn at a physiological concentration produces a long-lasting renal natriuresis, whereas previously reported natriuretic effects of UGn have required supraphysiological concentrations. Thus, our data point to proUGn as an endocrine (i.e. circulating) mediator of postprandial natriuresis, and suggest that the propeptide is secreted intact from the intestine into the circulation and processed to an active form at an extravascular site.

The nuclear receptor peroxisome proliferator-activated receptor ␣ (PPAR␣), in addition to regulating lipid homeostasis, controls the level of tissue damage after chemical or physical stress. To determine the role of PPAR␣ in oxidative stress responses, we examined damage after exposure to chemicals that increase oxidative stress in wild-type or PPAR␣-null mice. Primary hepatocytes from wild-type but not PPAR␣-null mice pretreated with the PPAR pan-agonist WY-14,643 (WY) were protected from damage to cadmium and paraquat. The livers from intact wild-type but not PPAR␣-null mice were more resistant to damage after carbon tetrachloride treatment. To determine the molecular basis of the protection by PPAR␣, we identified by transcript profiling genes whose expression was altered by a 7-day exposure to WY in wild-type and PPAR␣-null mice. Of the 815 genes regulated by WY in wild-type mice (p < 0.001; >1.5-fold or <؊1.5-fold), only two genes were regulated similarly by WY in PPAR␣-null mice. WY increased expression of stress modifier genes that maintain the health of the proteome, including those that prevent protein aggregation (heat stress-inducible chaperones) and eliminate damaged proteins (proteasome components). Although the induction of proteasomal genes significantly overlapped with those regulated by 1,2-dithiole-3-thione, an activator of oxidant-inducible Nrf2, WY increased expression of proteasomal genes independently of Nrf2. Thus, PPAR␣ controls the vast majority of gene expression changes after exposure to WY in the mouse liver and protects the liver from oxidant-induced damage, possibly through regulation of a distinct set of proteome maintenance genes.