Leukotriene B4 (LTB4) is a potent chemoattractant that is primarily involved in inflammation, immune responses and host defence against infection. LTB4 activates inflammatory cells by binding to its cell-surface receptor (BLTR). LTB4 can also bind and activate the intranudear transcription factor PPAR alpha, resulting in the activation of genes that terminate inflammatory processes. Here we report the cloning of the complementary DNA encoding a cell-surface LTB4 receptor that is highly expressed in human leukocytes. Using a subtraction strategy, we isolated two cDNA clones (HL-1 and HL-5) from retinoic acid-differentiated HL-60 cells. These two clones contain identical open reading frames encoding a protein of 352 amino acids and predicted to contain seven membrane-spanning domains, but different 5'-untranslated regions. Membrane fractions of Cos-7 cells transfected with an expression construct containing the open reading frame of HL-5 showed specific LTB4 binding, with a K(d) (0.154nM) comparable to that observed in retinoic acid-differentiated HL-60 cells. In CHO cells stably expressing this receptor, LTB4 induced increases in intracellular calcium, D-myo-inositol-1,4,5-triphosphate (InsP3) accumulation, and inhibition of adenylyl cyclase. Furthermore, CHO cells expressing exogenous BLTR showed marked chemotactic responses towards low concentrations of LTB4 in a pertussis-toxin-sensitive manner. Our findings, together with previous reports, show that LTB4 is a unique lipid mediator that interacts with both cell-surface and nuclear receptors.

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

The cDNA encoding a novel P 2 receptor was isolated from rat aortic smooth muscle cell library and functionally characterized. The cloned P 2 receptor exhibits structural features characteristic of the G protein-coupled receptor family and shows 44 and 38% amino acid identity with previously cloned rat P 2U and chicken P 2Y receptors, respectively. The cloned P 2 receptor is functionally coupled to phospholipase C but not to adenylate cyclase in C6 rat glioma cells transfected with the cloned P 2 expression vector. The rank order of agonist potency as judged by intracellular Ca 2؉ mobilization responses is UTP > ADP ‫؍‬ 2-methylthioATP > ADP␤S > ATP ‫؍‬ ATP␥S, which is not compatible with any of the previously characterized P 2 receptor subtypes. The nonselective P 2 antagonists, suramin and reactive blue-2, inhibit nucleotide-induced phospholipase C activation in cells expressing the cloned P 2 receptor. The cloned P 2 receptor mRNA is abundantly expressed in various rat tissues including lung, stomach, intestine, spleen, mesentery, heart, and, most prominently, aorta. The results indicate that the novel metabotropic P 2 receptor has pharmacological characteristics distinct from any of P 2 receptor subtypes thus far identified and suggest the existence of a novel regulatory system by extracellular nucleotides of potential significance. P 2 nucleotide receptors mediate a wide variety of physiological responses to extracellular nucleotides, including vascular smooth muscle contraction and relaxation, neurotransmission, and endocrine and exocrine secretion (1, 2). In the vascular system, nucleotides activate P 2 receptors on vascular smooth muscle cells to cause contraction (3). On the other hand, when nucleotides activate P 2 receptors on vascular endothelial cells, it stimulates release of the vasorelaxants, prostacyclin and NO, to cause vasorelaxation (4 -6). Nucleotide-induced vasoconstriction and relaxation were initially suggested to be mediated via two distinct subtypes of P 2 receptors, P 2X and P 2Y purinoceptors, respectively (7). However, recent studies on the agonist specificity and potency rank order have provided evidence for the existence of more than a single class of P 2 receptors in both vascular smooth muscle and endothelium. For example, the pyrimidine UTP as well as the P 2X selective agonist ␣,␤-methylene ATP evokes vasoconstriction in various vascular beds, leading to the suggestion that the third P 2 receptor that can interact with UTP, P 2U , mediates vasoconstriction, because UTP does not serve as a ligand for P 2X or P 2Y receptors (8 -12). Furthermore, activation of P 2 receptors with UTP and ␣,␤-methylene ATP in smooth muscle cells was demonstrated to lead to activation of different downstream effector molecules (i.e. phospholipase C (2) and a cation channel intrinsic to P 2X receptors (13), respectively). Several studies also demonstrated that depending on vascular beds and animal species, P 2U receptors mediate nucleotide-induced endothelium-dependent relaxation (11, 14, 15). However...

Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-Nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson’s disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging.