Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors.

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A3 receptors activation. We revealed that A3 receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A3 receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A3 receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A3 receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.

Adenosine modulates HIF-1{alpha}, VEGF, IL-8, and foam cell formation in a human model of hypoxic foam cells.

OBJECTIVE: Foam cell (FC) formation by oxidized low-density lipoprotein (oxLDL) accumulation in macrophages is crucial for development of atherosclerosis. Hypoxia has been demonstrated in atherosclerosis and hypoxia-inducible factor-1 (HIF-1) has been shown to promote intraplaque angiogenesis and FC development. As hypoxia induces HIF-1alpha stabilization and adenosine (ado) accumulation, we investigated whether this nucleoside regulates HIF-1alpha in FCs. METHODS AND RESULTS: Ado, under hypoxia, stimulates HIF-1alpha accumulation by activating all adenosine receptors (ARs). HIF-1alpha modulation involved extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38 MAPK), and protein kinase B (Akt) phosphorylation in the case of A(1), A(2A), A(2B), and ERK 1/2 phosphorylation in the case of A(3) receptors. Ado, through the activation of A(3) and A(2B) receptors, stimulates vascular endothelial growth factor (VEGF) secretion in a HIF-1alpha-dependent way. Furthermore, ado, through the A(2B) subtype, induces an increase of Interleukin-8 (IL-8) secretion in a ERK 1/2, p38, and Akt kinase-dependent but not HIF-1alpha-mediated way. Finally, ado stimulates FC formation, and this effect is strongly reduced by A(3) and A(2B) blockers and by HIF-1alpha silencing. CONCLUSIONS: This study provides the first evidence that A(3,) A(2B), or mixed A(3)/A(2B) antagonists may be useful to block important steps in the atherosclerotic plaque development ado-induced.

Thermodynamics of A2B adenosine receptor binding discriminates agonistic from antagonistic behaviour.

Thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees of the binding equilibrium of 12 ligands (six agonists and six antagonists) to the A(2B) adenosine receptor subtype have been determined by affinity measurements carried out on HEK 293 cells stably transfected with human A(2B) adenosine receptors at six different temperatures (4, 10, 15, 20, 25, 30 degrees C) and van't Hoff plot analysis have been performed. Affinity constants were obtained from saturation experiments of [(3)H]MRE 2029-F20 or by its displacement in inhibition assays for the other compounds. van't Hoff plots were essentially linear in the temperature range investigated, showing that the DeltaC(p) degrees of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 7< or =DeltaH degrees < or =23 kJ mol(-1)and 123< or =DeltaS degrees < or =219 JK(-1)mol(-1) for agonists and -40 < or =DeltaH degrees < or =-20 kJ mol(-1) and 10< or =DeltaS degrees < or =91 JK(-1)mol(-1) for antagonists indicating that agonistic binding is always totally entropy-driven while antagonistic binding is enthalpy and entropy-driven. In the -TDeltaS degrees versus DeltaH degrees plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated.

From tyrosine to glycine: synthesis and biological activity of potent antagonists of the purinergic P2X7 receptor.

The characterization of the native and recombinant P2X7 receptor continues to be hindered by the lack of specific and subtype-selective antagonists with a "druglike" profile. However, a tyrosine derivative named KN-62 exhibits selective P2X7 receptor-blocking properties. As a molecular simplification of KN-62, the present study was designed to evaluate the functional antagonistic properties of a novel series of glycine derivatives characterized by the presence of different phenyl-substituted piperazine moieties. Antagonistic activity of these glycine derivatives was tested on HEK293 cells transfected with the human P2X7 receptor. The most potent P2X7 receptor antagonist identified in this study (compound 4g) contains an o-fluorine substituent on the phenylpiperazine moiety and had an IC50 of 12.1 nM. The biological responses investigated were ATP-dependent Ca2+ influx across the plasma membrane and ethidium bromide uptake.

Adenosine and lymphocyte regulation.

Adenosine is a potent extracellular messenger that is produced in high concentrations under metabolically unfavourable conditions. Tissue hypoxia, consequent to a compromised cellular energy status, is followed by the enhanced breakdown of ATP leading to the release of adenosine. Through the interaction with A(2) and A(3) membrane receptors, adenosine is devoted to the restoration of tissue homeostasis, acting as a retaliatory metabolite. Several aspects of the immune response have to be taken into consideration and even though in general it is very important to dampen inflammation, in some circumstances, such as the case of cancer, it is also necessary to increase the activity of immune cells against pathogens. Therefore, adenosine receptors that are defined as "sensors" of metabolic changes in the local tissue environment may be very important targets for modulation of immune responses and drugs devoted to regulating the adenosinergic system are promising in different clinical situations.