Hepatic metabolic alterations in rats treated with low-dose endotoxin and aspirin: an animal model of Reye's syndrome.

The administration of a sublethal dose of endotoxin (LPS) followed one hour later by a low dose of aspirin (LPS + ASA) to fasted rats leads to biochemical perturbations similar to Reye's syndrome. In this study hepatic energy metabolism was assessed in freeze-clamped liver samples (12 hours posttreatment) obtained from (250 to 300 g Sprague-Dawley) rats. The administration of aspirin alone to fasted rats did not significantly alter the hepatic levels of adenine nucleotides, total ketones, or acyl-CoA thioesters as compared to controls. In contrast, in both LPS and LPS + ASA samples, there were declines in ATP/ADP ratio (P less than .005), total ketones (P less than .001) and acetyl CoA (P less than .005) compared to their respective controls. A striking alteration in acyl-CoA thioesters was observed in LPS + ASA-treated animals. Unlike control, aspirin, or LPS-treated animals, LPS + ASA-treated animals accumulated relatively large amounts of unusual CoA esters, including propionyl-CoA, (iso)butyryl-CoA, beta-methylcrotonyl-CoA, and isovaleryl-CoA, metabolites of the branch chain amino acid and odd-chain fatty acid oxidation pathways. The acyl-CoA profile is similar to that obtained in patients with Reye's syndrome. Like human patients with Reye's syndrome, these rats showed hyperammonemia, compromised fatty acid oxidation, and accumulation of branched chain amino acid oxidation metabolites. Accumulation of these intermediates with LPS + ASA is a possible mechanism for the potentiation of Reye's syndrome by aspirin. These findings provide biochemical evidence that sublethal doses of LPS + ASA administered to fasted rats produces an animal model of Reye's syndrome.

Quantitative analysis of neuropeptide Y receptor association with beta-arrestin2 measured by bimolecular fluorescence complementation.

BACKGROUND AND PURPOSE: beta-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and beta-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein-protein interactions. EXPERIMENTAL APPROACH: Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and beta-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor-beta-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/beta-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis. KEY RESULTS: NPY stimulation promoted association between Y1-Yc and beta-arrestin2-Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor-YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, beta-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for beta-arrestin2. Receptor mutagenesis combined with beta-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support beta-arrestin2 association, and that Y2 receptor-beta-arrestin2 interaction was enhanced by the intracellular loop mutation H155P. CONCLUSIONS AND IMPLICATIONS: The BiFC approach quantifies Y receptor ligand pharmacology focused on the beta-arrestin2 pathway, and provides insight into mechanisms of beta-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein-protein interaction.

Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation.

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Roles for beta II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells.

beta-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O(2)). Neutrophils and neutrophilic HL60 cells contain both betaI and betaII-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O(2) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of betaII-PKC from betaI-PKC null cells inhibited ligand-initiated O(2) generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), with betaII-PKC but not with RACK1, a binding protein for betaII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O(2) generation. Therefore, betaII-PKC but not betaI-PKC is essential for activation of O(2) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast, RACK1 is involved in negative signaling for O(2) generation. RACK1 binds to betaII-PKC but not with the p47(phox).betaII-PKC complex. RACK1 may divert betaII-PKC to other signaling pathways requiring beta-PKC for signal transduction. Alternatively, RACK1 may sequester betaII-PKC to down-regulate O(2) generation.

TNFalpha elicits association of PI 3-kinase with the p60TNFR and activation of PI 3-kinase in adherent neutrophils.

Tumor necrosis factor (TNFalpha) is an incomplete secretagogue in neutrophils and requires the engagement of beta integrins to trigger secretion of superoxide anion (O(-)(2)). The p60 TNF receptor (p60TNFR) is responsible for signal transduction for activation of O(-)(2) generation. Activation of TNFalpha-triggered O(-)(2) generation in neutrophils adherent to fibrinogen-coated surfaces involves the beta2 integrin receptor CD11b/CD18. Phosphoinositide 3-kinase (PI 3-kinase) is essential for activation of O(-)(2) generation; wortmannin, an inhibitor of PI 3-kinase, inhibited TNFalpha-elicited O(-)(2) generation. p60TNFR immunoprecipitated from neutrophils was associated with immunoreactivity to PI 3-kinase in adherent neutrophils exposed to TNFalpha, but not in TNFalpha-treated neutrophils in suspension. In addition, PI 3-kinase immunoprecipitated from TNFalpha-activated neutrophils showed enhanced activity in adherent but not in nonadherent neutrophils. These findings suggest that synergism between CD11b/CD18 and p60TNFR in the presence of TNFalpha is required to elicit assembly of a signaling complex involving association of p60TNFR with PI 3-kinase, activation of PI 3-kinase, and generation of O(-)(2).

Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells.

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete beta-PKC, both betaI- and betaII-PKC, but not alpha-PKC, delta-PKC, or zeta-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of beta-PKC in dHL60 cells elicited selective inhibition of O-2 generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited beta-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by beta-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in beta-PKC-depleted dHL60 cells. Phosphorylation of p47(phox) and translocation of p47(phox) to the membrane are essential for activation of the NADPH oxidase and generation of O-2. beta-PKC depletion had no effect on the level of p47(phox) in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47(phox) to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in beta-PKC-depleted cells. These results indicate that beta-PKC is essential for signaling for O-2 generation but not cell adherence or azurophil degranulation. Depletion of beta-PKC inhibited ligand-induced phosphorylation of p47(phox), translocation of p47(phox) to the membrane, and activation of O-2 generation.

Negative regulation of ligand-initiated Ca(2+) uptake by PKC-beta II in differentiated HL60 cells.

In phagocytic cells, fMet-Leu-Phe triggers phosphoinositide remodeling, activation of protein kinase C (PKC), release of intracellular Ca(2+) and uptake of extracellular Ca(2+). Uptake of extracellular Ca(2+) can be triggered by store-operated Ca(2+) channels (SOCC) and via a receptor-operated nonselective cation channel(s). In neutrophilic HL60 cells, the PKC activator phorbol myristate acetate (PMA) activates multiple PKC isotypes, PKC-alpha, PKC-beta, and PKC-delta, and inhibits ligand-initiated mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). Therefore PKC is a negative regulator at several points in Ca(2+) mobilization. In contrast, selective depletion of PKC-beta in HL60 cells by an antisense strategy enhanced fMet-Leu-Phe-initiated Ca(2+) uptake but not mobilization of intracellular Ca(2+). Thapsigargin-induced Ca(2+) uptake through SOCC was not affected by PKC-beta II depletion. Thus PKC-beta II is a selective negative regulator of Ca(2+) uptake but not release of intracellular Ca(2+) stores. PKC-beta II inhibits a receptor-operated cation or Ca(2+) channel, thus inhibiting ligand-initiated Ca(2+) uptake.

Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils.

Tumor necrosis factor-alpha (TNF-alpha) triggers degranulation and oxygen radical release in adherent neutrophils. The p60TNF receptor (p60TNFR) is responsible for proinflammatory signaling, and protein kinase C (PKC) is a candidate for the regulation of p60TNFR. Both TNF-alpha and the PKC-activator phorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR. Receptor phosphorylation was on both serine and threonine but not on tyrosine residues. The PKC-delta isotype is a candidate enzyme for serine phosphorylation of p60TNFR. Staurosporine and the PKC-delta inhibitor rottlerin inhibited TNF-alpha-triggered serine but not threonine phosphorylation. Serine phosphorylation was associated with receptor desensitization, as inhibition of PKC resulted in enhanced degranulation (elastase release). After neutrophil activation, PKC-delta was the only PKC isotype that associated with p60TNFR within the correct time frame for receptor phosphorylation. In vitro, only PKC-delta, but not the alpha-, betaI-, betaII-, or zeta-isotypes, was competent to phosphorylate the receptor, indicating that p60TNFR is a direct substrate for PKC-delta. These findings suggest a selective role for PKC-delta in negative regulation of the p60TNFR and of TNF-alpha-induced signaling.

Cromolyn inhibits assembly of the NADPH oxidase and superoxide anion generation by human neutrophils.

Early and late phase reactions have been observed in asthma; the late phase reaction is characterized by accumulation of inflammatory cells such as neutrophils. Activated neutrophils degranulate and assemble an active NADPH oxidase, which generates superoxide anion (O2-), reactions that have been implicated in lung tissue damage. Preincubation of neutrophils with the asthma drug cromolyn sodium selectively inhibited FMLP (10(-7) M) and PMA (0.1 microgram/ml) elicited O2- generation but not degranulation. To further characterize the mechanism of this inhibition we examined the effect of cromolyn on the NADPH oxidase complex and the signaling pathways for its assembly. Ca2+ mobilization and activation of protein kinase C have been implicated as signals for activation of the NADPH oxidase. Ca2+ mobilization triggered by FMLP was significantly decreased by 21.2% in cromolyn-treated cells. In contrast, cromolyn did not interfere with translocation or activity of protein kinase C. Membranes prepared from neutrophils stimulated with 0.5 microgram/ml PMA generated O2-, indicating assembly of an active NADPH oxidase; cromolyn did not inhibit this membrane-associated, preassembled oxidase. In contrast, preincubation of neutrophils with 100 microM cromolyn before addition of PMA decreased the capacity of the membranes to generate O2- by 57.3%. These results indicate that cromolyn inhibited the assembly of an active NADPH oxidase. The efficacy of cromolyn may be associated with inhibition of assembly of an active NADPH oxidase in the neutrophil and prevention of oxygen radical-induced tissue damage.