adenosine-5--(n-ethylcarboxamide) and Astrocytoma

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Topics: Adenosine; Adenosine A2 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Antineoplastic Agents; Astrocytes; Astrocytoma; Blotting, Western; Cell Growth Processes; Cell Line, Tumor; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunoprecipitation; Neuroprotective Agents; Phenethylamines; Purines; Pyrimidines; Radioligand Assay; Receptor, Adenosine A2B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Threonine; Time Factors; Transfection; Triazoles; Tumor Necrosis Factor-alpha

The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the levels of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A1 and A2 agonist 5'-(N-ethylcarboxamido) adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A1-specific agonists R-phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A2a-specific agonist CGS-21860 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A2a-selective antagonists 8-(3-chlorostyryl) caffeine and KF17837 [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine] , which have also some antagonistic properties at A2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A1 antagonist 8-cyclopentyl-1,3 dipropylxanthine is much less potent. The results indicate that adenosine A2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Antineoplastic Agents; Astrocytoma; Blotting, Northern; Caffeine; Dose-Response Relationship, Immunologic; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Purinergic P1 Receptor Agonists; Receptors, Purinergic P1; RNA, Messenger; Stereoisomerism; Tumor Cells, Cultured; Xanthines

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca2+ and a sustained inhibition of forskolin-, dopamine- and 5'-N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca2+]i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca2+ (10(-7) to 10(-3) M), both in ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca2+]i and subsequent entry of Ca2+ which directly interacts with a component of the adenylyl cyclase system.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Astrocytoma; Bradykinin; Brain Neoplasms; Calcimycin; Calcium; Calmodulin; Colforsin; Cyclic AMP; Dopamine; Egtazic Acid; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Neoplasm Proteins; Pyrrolidinones; Rolipram; Signal Transduction; Tumor Cells, Cultured

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Adenylyl Cyclases; Astrocytoma; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Histamine; Humans; Hydrolysis; Pertussis Toxin; Phosphatidylinositols; Receptors, Purinergic; Tumor Cells, Cultured; Type C Phospholipases; Vasodilator Agents; Virulence Factors, Bordetella